# The role of matrix proteins in eggshell formation

M.T. Hincke, Yves Y. Nys, Joël Gautron

► To cite this version:

M.T. Hincke, Yves Y. Nys, Joël Gautron. The role of matrix proteins in eggshell formation. The Journal of Poultry Science, 2010, 47 (3), pp.208-219. 10.2141/jpsa.009122. hal-02661283

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# The Role of Matrix Proteins in Eggshell Formation

Maxwell T. Hincke<fnref n="1" />, Yves Nys<fnref n="2" /> and Joel Gautron<fnref n="2" />

In this article we review the results of recent proteomic, transcriptomic and genomic analyses of the eggshell constituents and draw attention to the impact of this data on current understanding of eggshell mineralization. The most abundant constituents of the chicken eggshell have been identified. An exciting new approach is to determine which genes are upregulated during the onset of mineralization. New information from studies with purified native or recombinant eggshell proteins are necessary for *in vitro* tests to gain insight into the role of each isolated matrix component, and eventually to learn how they may function synergistically. One important goal will be to determine the impact and importance of post-translational modification of matrix components (glycosylation, glycanation, phosphorylation, etc.), which could greatly alter their properties and interactions. These investigations will continue to provide new insights into function of integrated defense strategies that operate at biomineralized barriers. Genes involved in the physical or chemical defense of the egg are functional candidates for marker assisted selection to improve egg and eggshell quality.

**Key words:** calcite, eggshell, osteopontin, Ovocalyxin, Ovocleidin

J. Poult. Sci., 47: 208-219, 2010

## 1. Introduction

The avian egg is a reproductive structure that protects the embryo from external physical and microbial attacks; at the same time this complex structure regulates the exchange of metabolic gases and water, and provides calcium to the developing embryo. The unfertilized egg is a nutritious foodstuff for human consumption and shell quality is of paramount importance. Alterations in egg-shell properties are directly related to increasing risk of egg contamination and food-borne outbreaks for the consumer. Many studies have been conducted on avian eggs and most have centered on the egg of the domestic chicken (Gallus gallus). This considerable body of work has provided insight into the structure and formation of the eggshell (Hincke et al., 2008a). In this article we review the results of recent proteomic, transcriptomic and genomic analyses of the eggshell constituents and draw attention to the impact of this data on current understanding of eggshell mineralization.

## 2. Eggshell Biosynthesis/Formation

Calcified matrices in vertebrate biology are biphasic composites that usually contain collagenous and non-

collagenous elements in intimate contact with mineral (Robey, 1996). While the avian eggshell is a complex and highly structured calcitic bioceramic with extensive inter-mingling of both its organic and inorganic phases, it also demonstrates a spatial separation between its organic framework and mineralized components, with a modest overlap between the eggshell membrane and calcified egg-shell (Arias et al., 1993; Dennis et al., 1996; Nys et al., 1999; Nys et al., 2004) (Fig. 1). During avian egg formation, it sequentially acquires all of its components as it passes through specialized regions of the oviduct. The egg is composed of a central yolk surrounded by the albumen, eggshell membranes, calcified eggshell and cuticle (Roberts, 2004).

Following ovulation, the yolk is captured by the infundibulum where the developing egg remains for about 15 minutes while the perivitelline membrane is formed. The yolk is the main source of energy and fat soluble vitamins for the developing embryo because it contains all lipidic components of the egg (Burley and Vadehra, 1989). During the following 3-4 hour period, in which the yolk/ovum complex travels down the largest portion of the oviduct, the magnum, it progressively acquires the albumen (Nys et al., 1999, 2004). The albumen is composed of water, salts and proteins that nourish the developing embryo and prevent the growth of micro-organisms. The alkaline pH of the albumen and the presence of proteins such as ovotransferrin and lysozyme significantly reduce the growth of micro-organisms

<footnote n="1">¹ Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, Canada</footnote>

<footnote n="2">² INRA, UR 83 Recherches avicoles, F-37380, Nouzilly, France</footnote>
<figure><img src="image_3.png" /><figcaption>Fig. 1. Scanning electron micrographs illustrating the morphology of the eggshell and eggshell membranes.</figcaption></figure>

A. Eggshell cross-fractured to reveal the shell membrane (SM), mammillary layer (ML) and palisade layer (PL); B. Higher magnification of the membrane - mammillary body interface. Outer shell membrane fibres (OSM) insert into the tips of the mammillary bodies (MB). Inner shell membranes (ISM); C. Enlargement of the shell membrane fibres (SMF) to reveal their interwoven and coalescing nature; D. Inner aspect of the inner shell membrane (ISM), demonstrating the limiting membrane (LM) that surrounds the egg white (here removed during sample preparation).

Scale bars: A, 50 mm; B, 20 mm; C and D, 2 mm.

Reprinted from Hincke *et al.*, 2000a, with permission from Elsevier.

(Deeming, 2002). The albumen also stabilizes the developing embryo within the fertilized egg. The combination of albumen and yolk provide a perfect balance of the nutrients needed for embryonic growth; the egg possesses very high nutritional value as a human foodstuff because

of its diverse and readily available components (Suess-Baum, 2007).

As the yolk and albumen complex next travel through the proximal (white) isthmus, the membranes are acquired in a 1 to 2 hour period. This meshwork of in-
In summary, matrix components play an active role in the control of calcite growth kinetics and crystal morphology during eggshell mineralization, and therefore regulate the textural properties and resulting biomechanical strength of the eggshell.

# 4. Eggshell Matrix Proteins

## 4.1 Proteomics

The eggshell mineral is associated with an organic matrix composed of proteins, glycoproteins and proteoglycans, termed “eggshell matrix proteins”, which are progressively incorporated from the precursor milieu (uterine fluid) during calcification. Their function is thought to influence the fabric of this biomaterial and/or to participate in its antimicrobial defenses. These non-mineral constituents represent about 2% by weight of the calcified eggshell, and can be released for study by demineralization of the eggshell by calcium chelation (EDTA or EGTA) or acid demineralization (acetic acid or HCl), yielding soluble and insoluble constituents. A complex array of distinct protein bands was demonstrated in the soluble intra- and extra-mineral compartments by 1 D-electrophoresis (SDS-PAGE) (Hincke et al., 1992; Gautron et al., 1996), and in the precursor uterine fluid, showing different patterns between the 3 stages of the eggshell calcification process (initial, growth and terminal) (Gautron et al., 1997). N-terminal sequencing of the electrophoretic bands allowed the egg white proteins ovalbumin, lysozyme and ovotransferrin to be identified (Hincke, 1995; Hincke et al., 2000; Gautron et al., 2001 b). N-terminal and internal amino acid sequencing of other protein bands revealed that they did not correspond to previously identified proteins and these have been subjected to more intensive investigation. Purification schemes using ion exchange (diethylaminoethyl (DEAE) - Sepharose and carboxymethyl (CM) - Sepharose) and hydroxyapatite were developed to isolate Ovocleidin-17 (Hincke et al., 1995) and Ovocalyxin-32 (Hincke et al., 2003) from eggshell extracts.

Ovocleidin-116 (OC-116) and Ovocalyxin-36 (OCX-36) were characterized by a combination of molecular cloning, immunochemistry and bioinformatics (Hincke et al., 1999; Gautron et al., 2007). Another associated approach was to compare the available Expression Sequence Tag (EST) sequences to partial protein or nucleotide sequences from egg components. This method was successfully used to characterize a 32 kDa band abundant in uterine fluid at the terminal phase of shell calcification (Ovocalyxin-32) (Gautron et al., 2001a). Such studies led to the concept that eggshell matrix protein components form three characteristic groups:

i) “Egg white” proteins which are also present in the eggshell - these include ovalbumin, the most abundant egg white protein (Hincke, 1995), lysozyme, an antimicrobial protein with hydrolytic activity against peptidoglycans on cell walls of Gram-positive bacteria (Hincke et al., 2000) and ovotransferrin, which sequesters iron necessary for

bacterial growth (Gautron et al., 2001b);

ii) Ubiquitous proteins that are found in many tissues - examples are osteopontin, a phosphorylated glycoprotein present in bone and other hard tissues of birds and mammals (Pines et al., 1994; Hincke and St. Maurice, 2000; Fernandez et al., 2003; Hincke et al., 2008 b; Chien et al., 2008, 2009), and clusterin, a widely distributed secretory glycoprotein that is also found in chicken egg white (Mann et al., 2003); and

iii) Eggshell-specific matrix proteins unique to the shell calcification process that are secreted by cells in specific regions of the oviduct where eggshell mineralization is initiated (red isthmus) and continues to completion (uterus). These matrix components are termed Ovocleidins (ovo, Latin - egg; kleidoun, Greek - to lock in, implying a functional role) or Ovocalyxins (ovo, Latin - egg; calyx, Latin - shell, referring to their shell location), with distinction based on apparent molecular weight by SDS-PAGE when initially characterized.

Recently, a high-throughput tandem-mass spectrometry approach (MS/MS) identified more than 500 eggshell matrix proteins (Mann et al., 2006), including the most abundant proteins that were already known (above). It is highly unlikely that all 520 proteins perform eggshell-specific functions or are involved in eggshell assembly. The majority of them are proposed to be remnants of previous stages of egg formation occurring in proximal segments of the oviduct, or intracellular proteins released by breakdown of the cells lining the oviduct during normal turnover (Mann et al., 2006). According to this hypothesis, all proteins that are present in the uterine fluid during the calcification process become assimilated into the eggshell, many of them in a non-specific manner.

Two possible roles for eggshell-specific matrix proteins have been proposed; both reflect the protective function of the eggshell in avian reproduction: regulation of eggshell mineralization and antimicrobial defence. Egg calcification occurs in three distinct phases (initiation, active calcification, and termination of shell calcification); each phase of shell mineralization is associated with a specific protein electrophoretic profile for the uterine fluid, suggesting that these molecules play specific roles during the calcification process (Gautron et al., 1997). The matrix proteins described in the next section are abundant components of the eggshell matrix and exhibit characteristics that are relevant to eggshell mineralization.

Ovocleidin-17 (OC-17) was the first eggshell-specific matrix protein to be isolated and characterized following its chromatographic purification after eggshell decalcification (Hincke et al., 1995). OC-17 is an abundant eggshell-matrix specific protein (40µg/g shell) (Mann et al., 2002). It is secreted by the tubular gland cells in the shell gland; within the shell it is distributed throughout the shell matrix, but concentrated in the mammillary bodies (Hincke et al., 1995). OC-17 is phosphorylated on two possible sites, Ser-61 and -67 (Mann and Siedler, 1999), and also can be glycosylated at Asn-59 to yield a 23 kDa
form: (Ovocleidin-23) (Mann, 1999). Glycosylation occurs at the N-glycosylation site consensus sequence, Asn-Ala-Ser which contains Ser-61. In the glycosylated form of the protein, Ser-61 is not phosphorylated, indicating that these modifications are mutually exclusive. In addition, peptides without modification at Asn-59 or Ser-61, with phosphorylation of Ser-67 only, or with no phosphorylation at all, have been detected (Mann *et al.*, 2007). The function of these modifications remains unknown but the phosphorylation sites are preserved in closely related eggshell proteins isolated from other avian species (see below) suggesting their importance.

Detailed studies have identified homologous eggshell matrix proteins in shell from other avian species. Comparison of their primary sequences revealed that ansocalcin (goose), struthiocalcin-1 & -2 (SCA-1 & -2, ostrich), dromaiocalcin-1 & -2 (DCA-1 & -2, emu) and rheocalcin-1 &-2 (RCA-1 & -2, rhea) form two groups based on sequence identity, serine phosphorylation and conservation of cysteine residues (Mann and Siedler, 2004; Lakshminarayanan et al., 2003; Mann and Siedler, 2006). Goose ansocalcin aligns reasonably well with proteins of group 1 (63-70% identity with SCA-1, DCA-1, RCA-1), but OC-17 has much less sequence identity with group 2 where it is placed (37-39% with SCA-2, DCA-2, RCA-2). It can be proposed that homologous proteins are found in the shells of all other bird species. Database searches with these eggshell protein sequences reveal that they belong to a heterogeneous group of proteins consisting of a single C-type lectin domain (CTL) and display sequence homology to members of this family such as mammalian Reg (Regenerating islet-derived) proteins, pancreatic stone protein (lithostathine), fish Type II antifreeze proteins and anticoagulant proteins from snake venom (Zelensky and Gready, 2005). The x-ray structure of OC-17 has been determined; it reveals a mixed alpha helix/beta sheet structure and verifies the C-type lectin-like domain (Reyes-Grajeda et al., 2002, 2004). Preliminary x-ray crystallographic studies of struthiocalcin-1 have been reported (Reyes-Grajeda et al., 2007).

The properties of purified OC-17 and its goose homolog (ansocalcin), and their influence upon calcite crystallization patterns have been investigated and compared (Lakshminarayanan et al., 2002, 2003, 2005; Reyes-Grajeda et al., 2004). Functionally, OC-17 and ansocalcin do not appear to be completely equivalent in their effect on calcite crystal growth *in vitro* (Lakshminarayanan et al., 2002; Reyes-Grajeda et al., 2004). Ansocalcin showed reversible concentration-dependent aggregation in solution, and was reported to induce pits on growing calcite rhombohedral faces at lower concentrations (<50 µg/ml) and to nucleate polycrystalline aggregates of calcite crystals at higher concentrations (Lakshminarayanan et al., 2003). Aggregated ansocalcin may act as a template for the nucleation of calcite crystal aggregates (Lakshminarayanan et al., 2002). However, under the same conditions, OC-17 was not observed to aggregate in

solution nor induce the nucleation of calcite aggregates. Nevertheless, under different experimental conditions, Reyes-Grajeda and coworkers reported that OC-17 could modify the crystalline habit of calcium carbonate and the pattern of crystal growth at concentrations of 5-200 µg/ml (Reyes-Grajeda et al., 2004). Ovocleidin-17 and SCA-1, but not SCA-2, are reported to interact directly with carbonate anion, as a potential mechanism accounting for different effects upon calcite nucleation and crystal growth (Marin-Garcia et al., 2008).

**Ovocleidin-116 (OC-116)** was the first eggshell matrix protein to be cloned, by expression screening a uterine library using an antibody raised to the abundant 116-kDa protein observed in hen uterine fluid during the active calcification phase of shell formation (Hincke *et al.*, 1999). OC-116 is the most abundant eggshell matrix protein, estimated at 80µg/g eggshell powder (Mann *et al.*, 2002). It is relatively eggshell specific; however, it is also present in young chick cortical bone, laying hen medullary bone and growth plate hypertrophic chondrocytes suggesting an additional role in bone mineralization (Horvat-Gordon *et al.*, 2008). The N-terminus of the mature protein and conceptual translation product from cDNA correspond to that previously reported for a 200 kDa eggshell matrix proteoglycan that is converted to 120 kDa by chondroitinase ABC treatment (Carrino *et al.*, 1997). Therefore, OC-116 is the core protein (predicted 75 kDa) corresponding to the doublet bands of an eggshell dermalan sulfate proteoglycan (116 kDa and 180 kDa). It is hypothesized that the 180 kDa form of OC-116 corresponds to the N-glycosylated core protein with attached glycoaminoglycans, while the 116 kDa form corresponds to the protein without glycoaminoglycans (Hincke *et al.*, 1999). Sequencing of peptides purified from protease-treated eggshell extract reveal that both predicted N-glycosylation sites are modified; however while Asn-62 is entirely glycosylated, Asn-293 is only marginally occupied (Mann *et al.*, 2002). Detailed analysis of the carbohydrate structures attached to Asn-62 revealed 17 different oligosaccharide structures (Nimtz *et al.*, 2004); on the other hand, glycoaminoglycans associated with OC-116 have not yet been characterized. OC-116 is phosphorylated to a variable and partial extent on at least 22 serine and threonine residues. Two sites that were frequently identified with different cleavage methods were Ser-444 and Thr-664 (Mann *et al.*, 2007).

Ultrastructural immunocytochemistry indicates that OC-116 is synthesized and secreted from the granular cells of the uterine epithelium, and is incorporated into, and widely distributed throughout, the palisade region of the calcified eggshell (Hincke *et al.*, 1999). Transmission electron microscopy (TEM) of the organic matrix of the avian eggshell reveals two structural features within the palisade layer; vesicular structures with electron-lucent cores intermingle between flocculent sheets of organic material. OC-116 is predominately associated with the periphery of the vesicular structures that probably corre-
spond to the walls of microvesicular holes (voids) in the calcitic eggshell (Hincke *et al.*, 1999). Such localization studies do not distinguish between the differentially phosphorylated, N-glycosylated or glycanated forms of OC-116, nor would possible differences in eggshell distribution between the 116 and 180 kDa forms be detected by this technique. Crystal growth studies have shown that pure glycoaminoglycans affect calcite morphology, leading to crystal elongation (Arias *et al.*, 2002), suggesting that the sulfated form of OC-116 could influence eggshell mineralization via electrostatic interactions. Single nucleotide polymorphisms (SNPs) in the OC-116 gene are significantly associated with eggshell elastic modulus and thickness and egg shape (Dunn *et al.*, 2008).

**Ovocalyxin-32 (OCX-32)** was originally identified as a 32 kDa uterine fluid protein that is abundant in the terminal phase of shell formation (Gautron *et al.*, 2001a; Hincke *et al.*, 2003). Sequencing of peptides derived from the purified protein allowed Expressed Sequence Tag sequences (EST's) to be identified that were assembled to yield a full-length composite sequence whose conceptual translation product contained the complete amino acid sequence of Ovocalyxin-32. Ovocalyxin-32 is expressed at high levels in the uterine and isthmus regions of the oviduct and is secreted by the surface epithelial cells that line the lumen (Gautron *et al.*, 2001a). In the eggshell, Ovocalyxin-32 localizes to the outer palisade layer, the vertical crystal layer, and the cuticle of the eggshell, in agreement with its demonstration by Western blotting at high levels in the uterine fluid during the termination phase of eggshell formation (Gautron *et al.*, 2001a; Hincke *et al.*, 2003; Miksik *et al.*, 2007). A study of eggshell phosphoproteins identified phosphorylation of Ovocalyxin-32 at serines and threonines between position 257 and 268, but exact sites were not determined (Mann *et al.*, 2007).

The timing of OCX-32 secretion into the uterine fluid has been interpreted to suggest that it plays a role in the termination of eggshell calcification (Gautron *et al.*, 1997). This hypothesis originated from the observations of morphological changes in calcite crystals by uterine fluid collected during the terminal phase of calcification and the location of OCX-32 in the mineral pellet after its precipitation with calcium carbonate *in vitro* from fresh uterine fluid (Dominquez-Vera *et al.*, 2000; Hernandez-Hernandez *et al.*, 2008).

**Ovocalyxin-36 (OCX-36)** is a prominent 36 kDa protein present in the uterine fluid collected during the active calcification stage of shell mineralization. The protein is only detected in the regions of the oviduct where eggshell formation takes place (isthmus and uterus). Moreover, the uterine OCX-36 message, quantified by real time RT-PCR, is strongly upregulated during eggshell calcification (Gautron *et al.*, 2007). OCX-36 localizes to the calcified eggshell (predominantly in the inner part of the shell), and is abundant in the shell membranes. OCX-36 protein sequence displays significant identity with mam-

malian proteins that are associated with the innate immune response, such as lipopolysaccharide-binding proteins (LBP), bactericidal permeability-increasing proteins (BPI) and palate, lung and nasal epithelium clone (Plunc) family proteins. These belong to a superfamily of proteins that are key components of the innate immune system and act as the first line of host defense (Bingle and Craven, 2004). LBP proteins initiate the inflammatory host response upon the detection of a pathogen (Shumann *et al.*, 1990). OCX-36 may therefore participate in natural defense mechanisms that keep the egg and oviduct free of pathogens.

**Osteopontin (OPN)** is a phosphoglycoprotein associated with normal and pathological calcium mineralization (McKee and Nanci, 1996). In the chicken, osteopontin is found in both bone and eggshell (Pines *et al.*, 1994; Hincke *et al.*, 2008b). The oviduct expression of osteopontin is entirely uterine-specific and is temporally associated with eggshell calcification through coupling of physical distension of the uterus to osteopontin gene expression (Lavelin *et al.*, 1998).

Localization studies show that OPN is concentrated in the palisades region of the eggshell (Fernandez *et al.*, 2003, Hincke *et al.*, 2008b; Chien *et al.*, 2009). Osteopontin exists as two to three predominant forms in both eggshell and bone, with an apparent size by SDS-PAGE ranging between 46 and 54 kDa (Hincke *et al.*, 2008b), indicating that bone and eggshell OPN differ in their posttranslational modifications. A number of phosphorylated residues in chicken eggshell and osteoblast OPN have been identified, which partially overlap between the two tissues (Salih *et al.*, 1997; Mann *et al.*, 2007). Dephosphorylation of eggshell OPN greatly diminishes its ability to inhibit precipitation of calcium carbonate from a supersaturated solution (Hincke and St. Maurice, 2000). Osteopontin is synthesized and secreted by the granular epithelial cells of the shell gland (Pines *et al.*, 1994; Fernandez *et al.*, 2003; Hincke *et al.*, 2008b). The granular cells also synthesize and secrete ovocleidin-116 (Hincke *et al.*, 1999), suggesting that this epithelial cell type plays a major role in secretion of the eggshell matrix.

After decalcification and processing of the eggshell for TEM and Scanning electron microscopy (SEM), an extensive organic matrix network is observed throughout all regions, which includes interconnected fibrous sheets, irregularly shaped aggregates, vesicular structures, protein films, and isolated protein fibers. OPN is associated with protein sheets in the highly mineralized palisades region, but not with the vesicular structures (Hincke *et al.*, 2008b; Chien *et al.*, 2008; 2009). The association of OPN with parallel sheets of matrix, and more diffusely with the {104} crystallographic faces of eggshell calcite, may function in regulating palisade growth by orienting calcite crystals and by regulating the speed of mineralization. The elongated calcite crystals in the palisades region tend to be preferentially orientated with the (001) planes parallel (c-axis perpendicular) to the shell surface, which
orients the {104} plane at 44° tangential to the surface (Silyn-Roberts and Sharp, 1986; Rodriguez-Navarro et al., 2002). The {104} calcite face is the natural cleavage plane, and specific osteopontin binding to this growing crystal face during mineralization could modify the resistance of the shell to fracture along this plane. The finding of an interaction between OPN and the {104} eggshell calcite faces was confirmed by *in vitro* studies of synthetic calcite growth where inhibition by added OPN was observed at the {104} faces (Chien et al., 2008).

Unusual patterns of uterine OPN expression may underlie certain defects in eggshell mineralization. In birds laying eggs with normal eggshells, OPN is expressed uniformly by all the epithelial cells facing the uterine lumen (Arazi et al., 2009). Decreased OPN gene expression is correlated with reduction in eggshell thickness that is observed after xenoestrogen treatment (Kamata et al., 2009). Moreover, xenoestrogen treatment resulted in thinning of the mammillary layer at the same site where OPN is localized (Hincke et al., 2008b; Kamata et al., 2009). Reduced or absent OPN expression in specific regions of the uterine luminal epithelium is correlated with eggshell defects such as corrugations, pimples and cracks (Arazi et al., 2009). A candidate gene association analysis with eggshell matrix genes recently revealed that OPN SNP's were associated with eggshell fracture toughness (Dunn et al., 2008).

## 4.2 Transcriptomics

There are few reports of global gene expression in chickens. Oviduct gene expression has been compared in mature and juvenile birds, in which 266 over-expressed genes were related to dramatic changes due to sexual maturity and the onset of egg production (Dunn et al., 2009). Included in this list of differentially expressed genes are a number with known involvement in shell gland function, such as ion transporters and shell matrix proteins (OC-116, OCX-36, OCX-32 and OCX-21). Another recent study has focused on the uterus (shell gland) during deposition of the eggshell to identify genes that are differentially expressed in the uterus during eggshell calcification (Jonchere et al., 2010). In this study gene expression in the uterus where the eggshell is formed was compared with two other segments of the oviduct (magnum, white isthmus) to detect genes encoding proteins involved in supplying mineral and organic precursors that participate in eggshell formation. Using this unique approach, differential expression of genes reveals specific functions of each specialized region that secrete egg components. More than 600 differentially expressed uterine transcripts were detected, corresponding to over 460 different genes. Gene Ontology (GO) analysis was used for interpretation of protein function. The most highly represented GO terms were related to genes encoding ion transport proteins, which provide eggshell mineral precursors (Nys et al., 1999; Bar, 2009).

Uterine expression of 52 previously characterized eggshell proteins was confirmed, as well as transcripts for

several new proteins not yet characterized in the eggshell. This limited number is partly due to the fact that some eggshell proteins are also expressed in other tissues along the oviduct. Consequently, these proteins are present in the eggshell, but not revealed by analysis of transcriptional data. Proteomic analysis allows minute amounts of biologically active proteins in tissue or fluid to be identified; the eggshell proteome contains a complex mixture of uterine-derived proteins including proteins derived from degraded cells or basement membranes and those derived from the proximal oviduct (i.e., egg white, egg yolk and vitelline membrane proteins) (Mann et al., 2006). The number of eggshell proteins identified by mass spectrometry (528 proteins) is 4-5 times greater than those found in other egg compartments (i.e., 148 proteins in egg white, 137 in the vitelline membrane and 316 in egg yolk) (D'Ambrosio et al., 2008; Farinazzo et al., 2009; Mann, 2007; Mann, 2008; Mann and Mann, 2008). Thus, it is likely that the eggshell also passively incorporates proteins from the proximal oviduct. Protein sequences derived from upregulated genes were examined for the presence of a signal peptide, in order to identify those that could be potentially secreted by uterine cells for deposition in the shell. Amongst the upregulated uterine transcripts, more than 50 corresponded to proteins with signal peptide sequences; these proteins were classified according to their proposed biological function in the eggshell. Proteins believed to be involved in the biomineralization of the shell, such as osteopontin and ovocleidin-116 (OC-116) were identified with this approach. The restricted uterine expression of other shell-specific proteins, ovocalyxin-36 (OCX-36) and ovocalyxin-21 (OCX-21), was also noted. OCX-21 contains a brichos domain and consequently, could play a role as molecular chaperone (Gautron and Nys, 2007). Upregulation was noted for transcripts encoding up to 10 calcium binding proteins, which could be deposited in the shell to interact with mineral during shell formation. In addition, a number of proteins involved in protein folding, which could play an important role in proper folding of eggshell matrix components, were also upregulated. Finally, this study also revealed upregulation of transcripts encoding proteins with antimicrobial properties that could play a role in the protection of the egg.

In summary, global gene expression profiling of the hen's oviduct during sexual maturation and eggshell formation have revealed a large number of differentially expressed genes (Dunn et al., 2009; Jonchere et al., 2010). Upregulation of genes coding for eggshell-specific matrix proteins (OC-116 and ovocalyxins) occurs during both of these physiologically distinct processes, while OPN expression is only upregulated during eggshell calcification. This approach complements earlier focused proteomic analysis of the eggshell (Mann et al., 2006; 2007) that revealed more than 500 eggshell proteins; however, less than 10% of the identified proteins were common to both strategies. The characterization of all proteins in the eggshell is a prerequisite for exploration of functional
properties and regulation of uterine proteins involved in fabrication of the eggshell. Additional biochemical studies are needed to confirm the biological activity of these putative proteins and to understand their roles in providing nutrients and protection for the developing embryo. Genes involved in the physical or chemical defense of the egg are functional candidates for a marker assisted selection to improve egg and eggshell quality. For example, the OCX-32 gene is expressed at higher levels in a low egg production strain (compared to a high production strain) of Taiwanese country chickens (Yang et al., 2007).

### 4.3 Genomics

The release of the chicken genome sequence in 2004 was a tremendous advance permitting molecular biology - based approaches to avian biology, physiology, breeding etc. (International Chicken Genome Sequencing Consortium, 2004). Inspection revealed that the chromosomal localization of OC-116 is adjacent to that of osteopontin on chromosome 4. These 2 genes are contiguous with other mineralization-specific genes (Bone Sialoprotein, Dentin Matrix Protein 1), that form the SIBLING (small integrin-binding ligand, N-linked glycoprotein) mineralization gene locus first reported in mammalian genomes. This correspondence reflects synteny between avian and mammalian genomes (Hincke et al., 2008a). Moreover, investigations into the evolutionary genetics of vertebrate tissue mineralization suggest that OC-116 and other SIBLING proteins are members of the secretory calcium-binding phosphoprotein (SCPP) family that functions in tetrapod mineralization (Kawasaki and Weiss, 2003; 2006). Based on its position within this gene locus, OC-116 is predicted to be the avian ortholog of mammalian MEPE (matrix extracellular phosphoglycoprotein), and its N-terminus displays about 30% identity with the *Homo sapiens* MEPE protein sequence (Hincke et al., 2008a). As expected, there is synteny between the two avian genomes that have been sequenced to date: *Gallus gallus* and *Taeniopygia guttata* (Zebra Finch) (Stapley et al., 2008). Further study of common features of different avian OC-116's should help to define its features that are important for mineralization.

Proteins originating from the SCPP genes have a common characteristic; they bind calcium ions via acidic amino acids such as Glu, Asp and phospho-Ser (Kawasaki and Weiss, 2003). One member, OPN, regulates calcification in vertebrate biominerals such as bone and teeth (McKee and Nanci, 1996). Eggshell OPN inhibits calcium carbonate precipitation (Hincke and St. Maurice, 2000), and interacts specifically with the {104} calcite crystal face that may regulate shell calcification (Chien et al., 2008; Hincke et al., 2008b). Another SIBLING member, Dentin Matrix Protein 1 (DMP1), is also an eggshell matrix protein (Mann et al., 2006; Horvat-Gordon et al., 2008) but its influence upon calcite crystallization is not yet known. Of these proteins, OC-116 is uniquely specialized as an avian member of this mineralization-

specific family, supporting the hypothesis that it has a unique role in calcitic mineralization of the avian shell.

A complementary approach to establish the role of matrix proteins in the variability of the eggshells physical and mechanical properties has been taken using genetic and genomic approaches. This study reveals a number of significant associations between alleles of certain candidate genes (OC-116 and Ovocalyxins, OPN, ovalbumin) and measurements of eggshell biomechanical properties (Dunn et al., 2008). Single nucleotide polymorphisms (SNPs) in the OC-116 gene are significantly associated with the eggshell elastic modulus and thickness, and egg shape, whereas OPN was associated with eggshell fracture toughness. OCX-32 SNPs were found to be significantly associated with mammillary layer thickness.

### 5. Conclusion/Perspectives

This article reviews the results of recent proteomic, transcriptomic and genomic analyses of the eggshell constituents and draws attention to the impact of this data on current understanding of eggshell mineralization. The majority of constituents of the chicken eggshell have been identified. An exciting new approach is to determine which genes are upregulated during the onset of mineralization. Future effort to compare and contrast the chicken eggshell matrix proteins with those of other avian eggshells will pay dividends to better determine the function of the eggshell matrix proteins in the eggshell. Two functional roles have been proposed: (i) regulation of eggshell mineralization and (ii) antimicrobial protection of the egg and its contents. New information from studies with purified native or recombinant eggshell proteins are necessary for *in vitro* tests to gain insight into the role of each isolated matrix component, and eventually to learn how they may function synergistically. One important goal will be to determine the impact and importance of posttranslational modification of matrix components (glycosylation, glycanation, phosphorylation, etc.), which could greatly alter their properties and interactions. These investigations will continue to provide new insights into function of integrated defense strategies that operate at biomineralized barriers.

### Acknowledgments

MTH thanks the National Science and Engineering Research Council of Canada (NSERC), Ontario Ministry for Agricultural, Farm and Rural Affairs (OMAFRA), the Poultry Industry Council (PIC) and the Advanced Foods & Materials Network (AFMNet) for research funding. YN and JG also gratefully acknowledge the European Community for its financial support to RESCAPE (RESCAPE Food CT 2006-036018) and SABRE (European Integrating project Cutting-Edge Genomics for Sustainable Animal Breeding), and the French National Research agency (ANR) for its support of the Biocristal project (Projet ANR-05-BLAN-0233).
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