Gene expression profiling to identify eggshell proteins involved in physical defense of the chicken egg

The chicken egg is formed in the hen's left ovary and oviduct. The ovary supports the accumulation of egg yolk proteins and maturation of the ovum (Figure 1A). After ovulation, the yolk enters the oviduct, where albumen, eggshell membranes and the eggshell are sequentially deposited in the different segments of the hen's reproductive tract (magnum, white isthmus and uterus, respectively) (Figure 1). The hen manufactures a cleidoic egg [1], which is a completely self-sufficient and aseptic biological package for the extra-uterine development of the avian embryo. This adaptation implies that the egg must contain all components required for the complete extra-uterine development of a fertilized ovum into a viable chick in 21 days. To ensure this dynamic challenge, the egg must possess a broad range of biological activities and natural defenses [2, 3]. The avian egg contains vitamins, minerals and proteins (albumen and yolk), yolk lipids and calcium salts (eggshell) necessary for the development of the embryo. Furthermore, the chicken and egg have been an important basic food for humans worldwide for millennia. The egg has a high nutritive value from a well-balanced source of amino acids that are easily assimilated [4]. When faced with physical and/or microbial aggression, the egg has two major defensive mechanisms--a chemical protection system composed of yolk, albumen and eggshell matrix proteins that provide antimicrobial protection [2, 3, 5, 6], and the intact eggshell that acts as a physical barrier to protect against bacterial invasion [6, 7].

The eggshell itself is a complex bioceramic material formed in the uterus (shell gland) segment of the chicken's oviduct. It consists of inner and outer eggshell membranes, an intermediate calcified zone composed of mammillary and palisade layers, and an outer cuticle layer (Figure 2). Organic components and ions required for eggshell mineralization are secreted by the uterus into the acellular milieu of uterine fluid, which bathes the egg during its 20 hour travel through the hen's oviduct. The eggshell is composed of calcium carbonate (polycrystalline calcite) deposited onto the eggshell membranes that are pervaded with organic matrix, which itself is a complex mixture of proteins, glycoproteins and proteoglycans [8, 9]. The organic matrix plays a major role in assembly of the bioceramic layer and in determination of its mechanical properties. Therefore, identification of the protein complement of the uterus is the first step toward a more complete understanding of the diverse biological functions of the avian eggshell.

Matrix proteins are traditionally studied using a variety of biochemical and molecular techniques. These classical approaches have allowed identification of ten proteins (Figure 2) that belong to three functional groups. Firstly, three egg white proteins, ovalbumin [10], lysozyme [11] and ovotransferrin [12] are found in the eggshell. Secondly, eggshell contain ubiquitous proteins including osteopontin, a phosphorylated glycoprotein present in bone and other hard tissues [13], and clusterin, a secretory glycoprotein that is also found in the egg white [14]. Thirdly, several matrix proteins are unique to shell calcification and only secreted in regions of the oviduct where eggshell calcification occurs. Ovocleidin-17 (OC-17) was the first eggshell protein purified from the shell [15]. This secretory protein (OC-17) is a C-type, lectin-like phosphoprotein [16] that occurs in glycosylated (23 kDa) and nonglycosylated (17 kDa) forms in the shell matrix [17]. Ovocleidin-116 (OC-116) was the first eggshell matrix protein to be cloned [18]. OC-116 forms the protein core of a 120-200 kDa dermatan sulfate proteoglycan called ovoglycan [19, 20], which is found throughout the compact calcified eggshell [18]. Ovocalyxin-32 (OCX-32), a 32 kDa uterine-specific protein, is concentrated in the outer calcified region and in the cuticle of the calcified shell [21]. Ovocalyxin-36 (OCX-36) is a 36 kDa protein found only in the shell gland (uterus) where eggshell calcification takes place [22]. Uterine OCX-36 message levels are strongly up-regulated during eggshell calcification. OCX-36 is predominantly localized in the inner part of the shell and homologous to innate immune response proteins [22]. Ovocalyxin-21 (OCX-21) is another eggshell specific protein that was recently cloned and characterized [8].

Although many major proteins in the egg have been identified, we need a more complete and detailed picture of the genes encoding all proteins required for eggshell formation. The availability of the chicken genome sequence [23] and recent development of high-throughput genomic and proteomic assays provide powerful tools for a more complete characterization of egg components [24]. A major advance in understanding the complex nature of the eggshell and its assembly in the hen's oviduct came from the work of Mann et al. [25, 26], who used a focused proteomics approach to identify 528 proteins contained within the eggshell.

The present study provides an original description of the oviduct transcriptome in the laying hen and a repertoire of functional genes that are highly expressed in the uterus during eggshell calcification. Our approach provides the first global description of highly expressed uterine genes and their putative secretory proteins that are deposited in the eggshell. These functional components ensure proper eggshell formation, which provides a natural physical barrier and robust antimicrobial protection for the developing chick embryo or the edible egg.

The eggshell is a sophisticated dynamic structure essential for successful reproduction of birds. Its architecture allows the diffusion of gases (O₂ and CO₂) between the developing embryo and the external environment. It also functions as a natural mechanical barrier to protect egg contents from the physical and microbial environment. Its integrity and strength is therefore critical for survival of the developing embryo and for consumers to ensure that table eggs are free of pathogens. The exceptional mechanical properties of the shell are the result of interactions between eggshell minerals (calcium carbonate) and organic macromolecules (proteins, glycoproteins and proteoglycans), which comprise the organic matrix, a key factor required for shell calcification [7, 9, 34]. Although the chicken eggshell is a very effective protective system, bacteria can penetrate the egg or enter the uterus via retrograde movement of fecal fluid from the cloaca prior to eggshell formation. Antimicrobial protection is a function that has been most commonly ascribed to numerous egg white proteins that possess antimicrobial properties [3, 35], although this role was also described for the eggshell matrix. Partially purified eggshell matrix exhibits antimicrobial activity against Pseudomonas aeruginosa, Staphylococcus aureus and Bacillus cereus [36], which cannot be solely explained by the presence of lysozyme [11], ovotransferrin [12] and ovocalyxin-36 [22]--three principal antimicrobial proteins identified in the eggshell. In such a context, the identification and characterization of organic matrix components has stimulated numerous studies recently reviewed [7, 34].

In the present study, we have used transcriptional profiling of the hen's oviduct to identify genes that are differentially expressed in the uterus during eggshell calcification. Egg proteins are sequentially deposited in the magnum, white isthmus and uterus as the forming egg passes through the hen's oviduct (Figures 1 and 2). The entire oviduct originates from the same population of cells [37], which specialize at sexual maturity into specific regions (magnum, isthmus and uterus) responsible for the deposition of egg white (magnum), eggshell membranes (white isthmus) and calcified shell (uterus) as the egg and its shell are formed. Consequently, the comparison of gene expression in the uterus where the eggshell is formed with two other segments of the oviduct (magnum or white isthmus) should reveal genes encoding proteins involved in supplying mineral and organic precursors that participate in eggshell formation. Using this unique approach, differential expression of genes should reveal specific functions of each specialized region that secrete egg components. Our study revealed a total of 605 highly expressed transcripts that correspond to 469 different genes (UniGene database) and 437 proteins. Forty-five transcripts have no match in nucleotide or protein databases and are considered as unknown genes present in the chicken genome.

Previous studies have shown that the organic matrix is made of unique proteins including ovocleidin-116 [18], ovocalyxin-36 [22], ovocalyxin-32 [21] and ovocalyxin-21 [8] (Figure 2). These four proteins are preferentially expressed in the uterus during eggshell calcification. A single cDNA insert corresponding to ovocalyxin-32 was present on our array but not expressed in the oviduct tissue. In contrast, the other three specific eggshell matrix genes were expressed only in the uterus as expected. Osteopontin (SPP1), a phosphorylated glycoprotein found in bone, kidney and various body secretions is over-expressed in epithelial cells of the uterus during eggshell calcification [13]. SPP1 was over expressed in the uterus in our microarray study as indicated by a 3.9- and 4.1-fold higher expression when compared to magnum and isthmus, respectively. Sixteen additional genes, over-expressed on microarrays were validated using qRT-PCR. Genes selected for qRT-PCR verification represent a wide range of fold differences (log2 ratios from 0.1 to 6.3) in uterine genes with low levels (10 to 41% higher), intermediate levels (52 to 100% higher), high levels (114% to 273% higher) and very abundant levels (up to 300% greater) in the uterus when compared to either the magnum or isthmus. From the 32 samples used in the microarray analysis, 31 laying hen oviduct samples were over-expressed in uterus. Only a single sample, corresponding to the lowest fold change (log2 ratio of 0.1), could not be validated by qRT-PCR.

There are few reports of global gene expression in chickens, while only one paper is related to the hen's reproductive tract [38], where oviduct gene expression was compared in mature versus juvenile birds using a custom 8 K cDNA microarray. Consequently, the over expressed genes were related to the dramatic changes due to the sexual maturity and the onset of egg production. In contrast, our samples were collected from mature hens during active calcification of the eggshell (i.e., 18 h post ovulation). Therefore, our transcriptional analysis was focused on the uterus (shell gland) during deposition of the eggshell. This approach allowed us to establish for the first time, the uterine transcriptome and 605 activated genes potentially related to eggshell deposition and associated cellular pathways. The functions of the 605 novel uterine transcripts were investigated using Gene Ontology (GO) annotation. The GO terms of the over-expressed genes in the uterus were compared to all GO terms represented on the 14 K array. The most over-represented proteins (GO terms) were related to ion transport which occurs during calcification [39, 40]. Our transcriptional analysis has confirmed proteins previously identified as transporters and revealed new ionic transporters involved in supply of minerals needed for building the eggshell (Jonchère et al., in preparation). In addition, a GO term revealed a higher expression of proteins involved in synaptic transmission (Table 1). This observation could be related with the activation of muscle contraction and mobility of the uterus during rotation of the egg to facilitate calcification and/or final expulsion of the completed egg [41]. Our study has also demonstrated high abundance of genes involved in protein synthesis during the eggshell formation.

The uterus synthesizes both intracellular and extracellular proteins, which are secreted into the uterine fluid where the mineralization takes place. We paid particular attention to the extracellular proteins, which form the eggshell matrix and consequently are suspected to be involved in mineralization or chemical protection of the egg. Our first approach was to compare proteins encoded by uterine genes with those identified by proteomics. Indeed, proteomics is an important high-throughput methodology, which enabled the identification of 528 proteins in the calcified eggshell [25, 26]. Our study confirmed uterine expression of 52 previously characterized eggshell proteins and 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, although but not revealed by our transcriptional analysis. The main advantage of the proteomics method is the ability to identify minute amounts of biologically active proteins in tissue or fluid. The eggshell proteome contains a complex mixture of uterine-derived proteins, additional proteins derived from degraded cells or basement membranes and those issued from the upper oviduct (i.e., egg white, egg yolk and vitelline membrane proteins) [25, 42]. 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) [43–47]. Consequently, it is likely that the eggshell also passively incorporates proteins produced in the upper oviduct. To determine which proteins are potentially secreted by uterine cells and then deposited in the shell, we examined the presence of a signal peptide in 437 protein sequences obtained from the 605 highly-expressed uterine transcripts. A total of 54 proteins with signal peptide sequences were identified using several protein-centric databases (UniprotKB database, InterPro functional domain annotations, PubMed publications) (Table 2). These proteins were classified according to their biological function in the eggshell. The first group contains proteins involved in the biomineralization of the shell. For example, osteopontin [secreted phosphoprotein 1(SPP1)] is a protein found in both bone and eggshell [13]. The role of SPP1 in mineralization of the chicken eggshell has been described in detail [34]. Abnormal expression of SPP1 in the shell gland (uterus) is related to abnormalities and cracks in the eggshell [48]. Also included are ovocleidin-116 (OC-116), ovocalyxin-36 (OCX-36) and ovocalyxin-21 (OCX-21), which are three eggshell matrix proteins specific to uterine tissue [8, 18, 22]. Their presence is unique to the calcified shell and their expression limited to the uterus. OCX-21 contains a brichos domain and consequently, could play a role as molecular chaperone. A similar role is also proposed for endoplasmin (ENPL), a protein from the heat shock protein 90 family. Chaperone proteins in uterine fluid could play an important role in proper folding of the eggshell matrix, which is the crucial template for eggshell calcification. Several additional proteins involved in protein folding were identified in the 54 proteins possessing a signal peptide sequences. Among these, four proteins [ICOS ligand (ICOSLG), neuroplastin (NPTN), beta 2-microglobulin (B2M), butyrophilin subfamily 1 member A1(BTN1A1)] were previously identified in eggshell proteomic survey [25]. These four proteins contain immunoglobulin-like (Ig-like) domains involved in cell-cell recognition, cell-surface receptors and immune responses [49]. The Ig-like domain is one of the most common protein modules found in a variety of mammalian proteins including sandwich-like proteins, which are crucial for protein folding and conformation [50]. Lysosomal alpha manosidase (MAN2B1) plays also a role in protein folding and it is the most abundant uterine gene revealed by our microarray analysis. In the recent eggshell proteome survey [25], five proteins correspond to MAN2B1. MAN2B1 is a glycoside hydrolase, that participates in the metabolism of glycoproteins, maturation of N-glycans and in protein folding [51]. Its role is related to calnexin (CANX), an acidic protein (pI = 4.46) also identified as a putative uterine secretatory proteins, which have not been previously found among eggshell proteins. CANX is a molecular chaperone, which assists in protein folding. CANX binds only glycoproteins that have been folded by enzyme (i.e., MAN2B1). Consequently, these two proteins could be involved in metabolism of glycoprotein and proteoglycan, which are part of the eggshell matrix and thought to interact with calcite crystals and influence the texture of the mineralized shell and its mechanical properties [7, 9, 52].

SLIT, an axon guidance molecule involved in the embryonic development [53] was identified among our 54 secreted proteins (Table 2) and in the earlier eggshell proteomic analysis [25]. SLIT2 encodes a large extracellular matrix protein composed of leucine rich repeat motifs, which provide a structural framework for protein-protein interactions. In addition, SLIT protein contains a domain corresponding to epidermal growth factor (EGF) with a repeat pattern involving a number of cysteine residues thought to be important for the three-dimensional structure of proteins. Consequently, we believe that SLIT might be involved in folding of the eggshell matrix. It is also notable that SLIT has a calcium-binding site at the N-terminus of EGF-like domains. Calcium-binding properties often are a prerequisite for matrix proteins involved in calcium biomineralization. Consequently, SLIT could interact with calcium to favor crystal nucleation and morphology of crystals by interacting with some crystal faces of calcite. The ordered deposition of calcium carbonate (under the control of organic matrices) determines the texture of biominerals found in a large variety of calcified structures [39, 54]. Amongst the 54 putative secretatory proteins, we have identified ten additional calcium-binding proteins; some of them were not previously characterized in the eggshell. These calcium binding proteins are endoplasmin (ENPL), SLIT2, SLIT3 (described above), nucleobindin-2 (NUCB2), follistatin-related protein-1 (FSTL1) and FK506-binding protein 9 (FKBP9); all contain calcium-binding EF-hand domains. Calcium is also a ligand of Calsyntherin-3(CLSTN3) and mannose-binding protein C (MBL2), which could also interact with calcium during eggshell fabrication. Another interesting secretatory protein is podocalyxin (PODXL), a sialoprotein, which was first identified in the renal glomerular podocytes [55] and more recently as a selectin ligand that facilitates metastasis [56]. Because of its high net negative charge, PODXL could interact with calcium carbonate during the calcification of the eggshell.

We also identified dentin matrix protein-4 (DMP4) as a secreted uterine protein. DMP4 is a calcium-binding protein that plays a role in dentin mineralization. This protein is a member of the FAM20 family corresponding to secreted proteins that regulate differentiation and function of hematopoiesis cells [57]. This protein was predicted as secreted and was found in the recent proteome survey [25]. We also paid a particular attention to BMP-binding endothelial regulator protein (BMPER) and chordin (CHRD). BMPER is a secreted protein known to interact with bone morphogenetic proteins (BMP-2, -4 and -6) and BMP2/4 antagonists in humans [58, 59]. CHRD was first identified for its involvement in dorsalization of tissue in embryos. It is also a secreted protein, which binds BMP-2 -4 and -7 [60]. BMPs are members of the TGF-β superfamily of proteins and are known to induce the formation of new cartilage and bone following its ectopic implantation [61]. Studies in mollusks and coral suggest a role of BMPs in biomineralization [62–65]. Although BMP2 and BMP4 cDNAs were not present on our microarray, we used qRT- PCR to show higher level of expression (P < 0.02) of BMP2 in the uterus (0.686 ± 1.18) when compared to the magnum (0.034 ± 0.02). Therefore, it is likely that BMP2 is present in the uterine fluid and contributes to eggshell formation.

The second group of proteins, secreted in the uterus with a putative protective role, has antimicrobial properties. Antimicrobial proteins are found in the various compartments of the egg (yolk, egg white and shell), where they protect the egg against bacterial invasion, keeping the egg free of pathogens. Previous studies have shown that the eggshell matrix exhibits antimicrobial activity [36]. Three antimicrobial proteins (lysozyme, ovotransferrin and ovocalyxin-36) have been identified in the eggshell [11, 12, 22]. Our study has identified additional antimicrobial proteins secreted by the uterus, particularly proteins that contain Ig-like domains [ICOS ligand (ICOSL), neuroplastin (NPTN), beta-2-microglobulin (B2M), butyrophilin subfamily 1 member A1 (BTN1A1)], which are related to the immune responses [49]. Of particular interest are amyloid beta A4 protein (APP) and beta-amyloid protein 751 isoform (APP-751), which contain an amyloid extracellular domain and a heparin-binding domain. Heparin-binding proteins have basic domains, which might antimicrobial by binding to lipolysachharide (LPS) [66].

Our study has also revealed over-expression of avian β-defensin 9 (AvBD9) [previously called either gallinacin 9 or gallinacin 6] in the uterus. The avian β-defensins (AvBDs) are small cationic non-glycosylated peptides (1-10 kDa) with a three-stranded β-sheet structure connected with a β-hairpin loop that protect against gram-positive and gram-negative bacteria [5, 67]. In mammals, β-defensins are involved in innate immunity and are capable of evading pathogen resistance mechanisms. In birds, AvBD9 is highly expressed in the trachea, esophagus and crop, while lower expression is found in skin, liver, testis and vas deferens [67]. Our transcriptional analysis indicates that AvBD9 is also expressed in the chicken uterus, where this antimicrobial peptide could contribute to the aseptic environment of the hen's oviduct. This idea is supported by the appearance of AvBD1-3 in cultured vaginal cells following Salmonella enteritidis or LPS exposure [68].

The third group of candidate proteins is proteases and antiproteases, which are involved in blood coagulation, cell migration and proliferation, innate defense and gamete maturation. We have identified three proteases: cathepsin A (CTSA, a serine carboxypeptidase), glioma pathogenesis-related protein 1 (GLIPR1, which contains a calcium chelating serine protease domain) and beta-secretase 2 (BACE1, an aspartyl protease). Previous work has shown that proteolytic activity present in uterine fluid varies according to the stage of the calcification [69]. Proteases could have a specific and controlled role during the calcification process, by either degrading proteins or regulating processing of proteins into mature forms. For example, CTSA has important roles in protein catabolism and in posttranslational processing of proteins and peptides, which ensures their stability and proper maturation [70].

Seven over-expressed genes encoding uterine antiproteases were identified in our study. Amyloid beta A4 protein (APP), follistatin-related protein 1(FSTL1), tissue factor pathway inhibitor 2 (TFPI2) and beta-amyloid protein 751 isoform (APP-751), all contain a Kunitz/Bovine pancreatic trypsin inhibitor domain. Alpha2-antiplasmin (SERPINF2) belongs to the serine protease inhibitor (or serpin) family. BMP-binding endothelial regulator protein (BMPER) contains a trypsin inhibitor like cysteine rich domain; and tissue metalloproteinase inhibitor 2 (TIMP2) belongs to the tissue inhibitor of metalloproteinase (TIMP) family. Proteases inhibitors could locally regulate the proteolytic activity of the uterine proteases or have an antimicrobial action by inhibiting bacterial proteases [71]. Besides their potential role in physical and chemical defense of the egg, the proteases and anti-proteases are likely to participate in embryonic development. The embryo gradually mobilizes calcium from the eggshell to ensure bone formation; therefore, active release of proteases or anti-proteases is needed for normal development. Interestingly, several proteases and anti-proteases identified in our work (i.e., APP, BACE1 and possibly APP-751) have been described in other species as major agents of neurite outgrowth and cell survival [72], whereas SERPINF2, TIMP2 and TFPI2 are implicated in angiogenesis and morphogenesis [73–75]. Additionally, FSTL1 is a regulator of early mesoderm patterning, somitogenesis, myogenesis and neural development in the chick embryo [76].

Global gene expression profiling of the hen's oviduct during eggshell formation has revealed a large number of differentially expressed genes. Our study took advantage of tissue sampling from specialized segments of the oviduct that sequentially form different egg components and a bioinformatic analysis of the differentially expressed genes and their encoded proteins. This transcriptome approach enabled identification of more than 400 over-expressed genes in the uterus that are involved in providing precursors of the eggshell or proteins secreted into uterine fluid for fabrication of the eggshell and chemical protection of the egg. Our approach complements earlier focused proteomic analysis of the eggshell [25, 26] that revealed more than 500 eggshell proteins, albeit 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. Our study could lead to improvements in the hygienic quality of this important human food and reveal novel proteins that might be useful for pharmacological applications. In addition, genes involved in the physical or chemical defense of the egg against pathological agents, are functional candidates for a marker assisted selection to improve egg and eggshell quality. Furthermore, identification of all protein components in the egg will allow optimization of the egg's defense system and, consequently, contribute to reduce risk of food-borne diseases.