Proteomic analysis of quail calcified eggshell matrix: a comparison to chicken and turkey eggshell proteomes

Background: Eggshell mineralization in commercially important species such as chicken, turkey or quail is of interest as a general model of calcium carbonate biomineralization. Knowledge of proteins and molecular mechanisms in eggshell assembly may also pave the way to manipulation of thickness of the calcified layer or other features. Comparison of eggshell matrix proteomes of different species may contribute to a better understanding of the mineralization process. The recent publication of the quail genome sequence now enables the proteomic analysis of the quail shell matrix and this comparison with those of chicken and turkey. Results: The quail eggshell proteome comprised 622 identified proteins, 311 of which were shared with chicken and turkey eggshell proteomes. Forty-eight major proteins (iBAQ-derived abundance higher than 0.1 % of total identified proteome) together covered 94 % of total proteome mass. Fifteen of these are also among the most abundant proteins in chicken and turkey eggshell matrix. Only three proteins with a percentage higher than 1.0 % of the total had not previously been identified as eggshell matrix proteins. These were an uncharacterized member of the latexin family, an uncharacterized protease inhibitor containing a Kunitz domain, and gastric intrinsic factor. The most abundant proteins were ovocleidin-116, ovalbumin and ovocalyxin-36 representing approximately 31, 13 and 8 % of the total identified proteome, respectively. The major phosphoproteins were ovocleidin-116 and osteopontin. While osteopontin phosphorylation sites were predominantly conserved between chicken and quail sequences, conservation was less in ovocleidin-116. Conclusions: Ovocleidin-116 and ovocalyxin-36 are among the most abundant eggshell matrix proteins in all three species of the family Phasianidae analyzed so far, indicating that their presently unknown function is essential for eggshell mineralization. Evidence for other chicken eggshell-specific proteins in quail was inconclusive. Therefore measurement of additional eggshell proteomes, especially from species of different families and preferentially from outside the order Galliformes, will be necessary

Genetic variation in eggshell crystal size and orientation is large and these traits are correlated with shell thickness and are associated with eggshell matrix protein markers

The size and orientation of calcium carbonate crystals influence the structure and strength of the eggshells of chickens. In this study, estimates of heritability were found to be high (0.6) for crystal size and moderate (0.3) for crystal orientation. There was a strong positive correlation (0.65) for crystal size and orientation with the thickness of the shell and, in particular, with the thickness of the mammillary layer. Correlations with shell breaking strength were positive but with a high standard error. This was contrary to expectations, as in man-made materials smaller crystals would be stronger. We believe the results of this study support the hypothesis that the structural organization of shell, and in particular the mammillary layer, is influenced by crystal size and orientation, especially during the initial phase of calcification. Genetic associations for crystal measurements were observed between haplotype blocks or individual markers for a number of eggshell matrix proteins. Ovalbumin and ovotransferrin (LTF) markers for example were associated with crystal size, while ovocleidin-116 and ovocalyxin-32 (RARRES1) markers were associated with crystal orientation. The location of these proteins in the eggshell is consistent with different phases of the shell-formation process. In conclusion, the variability of crystal size, and to a lesser extent orientation, appears to have a large genetic component, and the formation of calcite crystals are intimately related to the ultrastructure of the eggshell. Moreover, this study also provides evidence that proteins in the shell influence the variability of crystal traits and, in turn, the shell’s thickness profile. The crystal measurements and/or the associated genetic markers may therefore prove to be useful in selection programs to improve eggshell quality

Avian eggshell biomineralization: an update on its structure, mineralogy and protein tool kit

The avian eggshell is a natural protective envelope that relies on the phenomenon of biomineralization for its formation. The shell is made of calcium carbonate in the form of calcite, which contains hundreds of proteins that interact with the mineral phase controlling its formation and structural organization, and thus determine the mechanical properties of the mature biomaterial. We describe its mineralogy, structure and the regulatory interactions that integrate the mineral and organic constituents. We underline recent evidence for vesicular transfer of amorphous calcium carbonate (ACC), as a new pathway to ensure the active and continuous supply of the ions necessary for shell mineralization. Currently more than 900 proteins and thousands of upregulated transcripts have been identified during chicken eggshell formation. Bioinformatic predictions address their functionality during the biomineralization process. In addition, we describe matrix protein quantification to understand their role during the key spatially- and temporally- regulated events of shell mineralization. Finally, we propose an updated scheme with a global scenario encompassing the mechanisms of avian eggshell mineralization. With this large dataset at hand, it should now be possible to determine specific motifs, domains or proteins and peptide sequences that perform a critical function during avian eggshell biomineralization. The integration of this insight with genomic data (nonsynonymous single nucleotide polymorphisms) and precise phenotyping (shell biomechanical parameters) on pure selected lines will lead to consistently better-quality eggshell characteristics for improved food safety. This information will also address the question of how the evolutionary-optimized chicken eggshell matrix proteins affect and regulate calcium carbonate mineralization as a good example of biomimetic and bio-inspired material design.French National Research Agency (ANR) European Commission ANR-13-BSV-0007-01Natural Sciences and Engineering Research Council of Canada (NSERC) RGPIN-2016-04410Le STUDIUMCentre Val de Loire, Nouzilly, France - University of Tour

The proteome of the calcified layer organic matrix of turkey (Meleagris gallopavo) eggshell

Background: Chicken eggshell mineralization is a prominent model for biomineralization not only because of its importance for avian reproduction but also because of the commercial interest associated with eggshell quality. An analysis and comparison of the protein constituents of eggshells of several species would contribute to a better understanding of the shell mineralization process. The recent publication of the turkey genome sequence now provides a basis for the in-depth analysis of the turkey eggshell proteome. Results: Proteomic analysis of turkey acid-soluble and acid-insoluble organic eggshell matrix yielded 697 identified proteins/protein groups. However, intensity-based absolute quantification (iBAQ) results indicated that the 47 most abundant identified proteins already constituted 95% of the total turkey eggshell matrix proteome. Forty-four of these proteins were also identified in chicken eggshell matrix previously. Despite these similarities there were important and unexpected differences. While ovocleidin-116 and ovocalyxin-36 were major proteins constituting approximately 37% of the identified proteome, other members of the group of so-called eggshell-specific proteins were not identified. Thus ovocalyxin-21 and ovocalyxin-32 were missing among matrix proteins. Conversely, major turkey eggshell proteins were not detected in chicken, such as the bone protein periostin, the mammalian counterpart of which is involved in many aspects of bone metabolism and which represented 10-11% of the total identified proteome. Conclusions: Even members of the same avian family show important differences in eggshell matrix composition and more studies on the proteome and the transcriptome level will be necessary to identify a common toolkit of eggshell mineralization and to work out species differences among functional eggshell protein sets and their role in eggshell production

Can museum egg specimens be used for proteomic analyses?

Extent: 5p.Background: Mass spectrometry and proteomic analyses have become powerful tools for the analysis of proteins and peptides. Investigation of proteins contained in the various layers of the avian eggshell has focused entirely on domesticated species. It has been widely assumed that this existing research can inform the study of wild bird species despite the fact that the vast majority of the diversity in avian species (~95%) exists outside the Orders to which domestic and poultry species belong. Museum collections offer a potentially valuable source of material for studying composition of wild avian eggshell matrix proteins. We used museum and fresh eggshells of common quails Coturnix coturnix to compare the protein composition of their organic matrices. Four eggs of domestic chickens were analysed simultaneously as a control for comparison to the fresh and museum quail eggs. The determination of the proteins was carried out using enzymatic cleavage followed by high-performance mass spectrometry. Results: We found that some of the expected key eggshell proteins (3 out of 11) were not present in the samples of museum quail egg. These proteins were either entirely absent from the museum eggs or the technique was unable to detect them. There was no pattern in the absent proteins in the sense of protein function or where they are located within the eggshell. Conclusion: We conclude it is likely that such studies on museum specimens using a proteomic approach will be limited in coverage of proteins and may, therefore, be misleading.Steven J Portugal, Helen J Cooper, Cleidiane G Zampronio, Laine L Wallace, Phillip Casse

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

<p>Abstract</p> <p>Background</p> <p>As uricoletic animals, chickens produce cleidoic eggs, which are self-contained bacteria-resistant biological packages for extra-uterine development of the chick embryo. The eggshell constitutes a natural physical barrier against bacterial penetration if it forms correctly and remains intact. The eggshell's remarkable mechanical properties are due to interactions among mineral components and the organic matrix proteins. The purpose of our study was to identify novel eggshell proteins by examining the transcriptome of the uterus during calcification of the eggshell. An extensive bioinformatic analysis on genes over-expressed in the uterus allowed us to identify novel eggshell proteins that contribute to the egg's natural defenses.</p> <p>Results</p> <p>Our 14 K Del-Mar Chicken Integrated Systems microarray was used for transcriptional profiling in the hen's uterus during eggshell deposition. A total of 605 transcripts were over-expressed in the uterus compared with the magnum or white isthmus across a wide range of abundance (1.1- to 79.4-fold difference). The 605 highly-expressed uterine transcripts correspond to 469 unique genes, which encode 437 different proteins. Gene Ontology (GO) analysis was used for interpretation of protein function. The most over-represented GO terms are related to genes encoding ion transport proteins, which provide eggshell mineral precursors. Signal peptide sequence was found for 54 putative proteins secreted by the uterus during eggshell formation. Many functional proteins are involved in calcium binding or biomineralization--prerequisites for interacting with the mineral phase during eggshell fabrication. While another large group of proteins could be involved in proper folding of the eggshell matrix. Many secreted uterine proteins possess antibacterial properties, which would protect the egg against microbial invasion. A final group includes proteases and protease inhibitors that regulate protein activity in the acellular uterine fluid where eggshell formation takes place.</p> <p>Conclusions</p> <p>Our original study provides the first detailed description of the chicken uterus transcriptome during formation of the eggshell. We have discovered a cache of about 600 functional genes and identified a large number of encoded proteins secreted into uterine fluid for fabrication of the eggshell and chemical protection of the egg. Some of these uterine genes could prove useful as biological markers for genetic improvement of phenotypic traits (i.e., egg and eggshell quality).</p

Microfocus X-ray scattering investigations of eggshell nanotexture

The avian eggshell is a highly ordered calcitic bioceramic composite, with both inorganic and organic constituents. The interactions between the inorganic and organic components within the structure are poorly understood but are likely to occur at the nanometre level. Thus structural variation at this level may impinge on the overall structural integrity and mechanical performance of the eggshell, and therefore analysis at this level is fundamental in fully understanding this ordered structure. In this study, structural changes in the mineral crystallites were investigated by microfocus small-angle X-ray scattering (µSAXS) using synchrotron radiation. Small-angle X-ray scattering (SAXS) can be used to investigate structures on the nanometre scale such as size, shape, arrangement and internal porosity. A microfocused X-ray beam, 1.5 µm vertically by 7 µm, was used to produce vertical linear scans of the eggshell section. SAXS patterns were taken from the eggshell membrane (inner surface of the eggshell) to the cuticle (outer surface of the eggshell). This allowed textural variations within the eggshell to be mapped. The scattering intensity profile was then used to derive the dimension of scattering objects that define the nanotexture. The nanotexture observed may result from the presence of the organic matrix, which is embedded as intracrystalline particles producing voids within the calcified framework of large (&gt;1 µm) calcite crystals. Porod analysis revealed the average size of a scattering interface to be approximately 4.5 nm with small changes that had a depth-dependent variation. These were largest at the mammillary layer/membrane boundary. The palisade layer displayed a small upward trend in size of scattering object. Parallel scans showed that the textural variations observed within the palisade layer are significant and indicate local subtextures. In addition, many of the patterns exhibit diffuse scattering streaks that could result from reflectivity from the larger crystallite interfaces. Changes in the orientation of diffuse streaks were observed within the different layers, the membranes, mammillary layer, palisade layer, vertical crystal layer and cuticle, indicating certain preferred orientations of the crystallites within the layers. The nanotextural variations that are apparent could have implications at the macroscopic level of the resulting eggshell

Zooarchaeological evidence for the exploitation of birds in medieval and early modern Estonia (ca 1200–1800)

In this paper, we discuss bird bones from sixteen sites across Estonia, focusing on the Medieval and Early Modern Period (ca 1200â1800). Zooarchaeology, stable isotope analysis and Zooarchaeology by Mass Spectometry (ZooMS) are used to explore how the exploitation of birds has differed between sites of various functions and locations. The results demonstrate the ubiquity of the chicken as the most abundant avian species in most sites and periods under study. The goose and the duck were the second and third most common species identified in the assemblages. Species diversity was highest at castle sites, where the presence of different wild birds can be associated with higher social status; however, the use of several bird species is unlikely to be food-related. The most frequently discovered wild birds were the black grouse and the western capercaillie, which are also known to have been served at feasts. This paper presents the first comprehensive study of Estonian avian zooarchaeological material from various contexts, giving a better overview of the importance of birds to historical communities