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

ABSTRACT: BACKGROUND: 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. RESULTS: 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. CONCLUSIONS: 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).

SUBMITTER: Jonchere V

PROVIDER: S-EPMC2827412 | biostudies-literature | 2010

REPOSITORIES: biostudies-literature

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

Jonchère Vincent V Réhault-Godbert Sophie S Hennequet-Antier Christelle C Cabau Cédric C Sibut Vonick V Cogburn Larry A LA Nys Yves Y Gautron Joel J

BMC genomics 20100121

<h4>Background</h4>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 p ...[more]

PMID: 20092629

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Project description:The avian eggshell membranes surround the egg white and provide a structural foundation for calcification of the eggshell which is essential for avian reproduction; moreover, it is also a natural biomaterial with many potential industrial and biomedical applications. Due to the insoluble and stable nature of the eggshell membrane fibres, their formation and protein constituents remain poorly characterized. The purpose of this study was to identify genes encoding eggshell membrane proteins, particularly those responsible for its structural features, by analyzing the transcriptome of the white isthmus segment of the oviduct, which is the specialized region responsible for the fabrication of the membrane fibres.The Del-Mar 14 K chicken microarray was used to investigate up-regulated expression of transcripts in the white isthmus (WI) compared with the adjacent magnum (Ma) and uterine (Ut) segments of the hen oviduct. Analysis revealed 135 clones hybridizing to over-expressed transcripts (WI/Ma?+?WI/Ut), and corresponding to 107 NCBI annotated non-redundant Gallus gallus gene IDs. This combined analysis revealed that the structural proteins highly over-expressed in the white isthmus include collagen X (COL10A1), fibrillin-1 (FBN1) and cysteine rich eggshell membrane protein (CREMP). These results validate previous proteomics studies which have identified collagen X (?-1) and CREMP in soluble eggshell extracts. Genes encoding collagen-processing enzymes such as lysyl oxidase homologs 1, 2 and 3 (LOXL1, LOXL2 and LOXL3), prolyl 4 hydroxylase subunit ?-2 and beta polypeptide (P4HA2 and P4HB) as well as peptidyl-prolyl cis-trans isomerase C (PPIC) were also over-expressed. Additionally, genes encoding proteins known to regulate disulfide cross-linking, including sulfhydryl oxidase (QSOX1) and thioredoxin (TXN), were identified which suggests that coordinated up-regulation of genes in the white isthmus is associated with eggshell membrane fibre formation.The present study has identified genes associated with the processing of collagen, other structural proteins, and disulfide-mediated cross-linking during eggshell membrane formation in the white isthmus. Identification of these genes will provide new insight into eggshell membrane structure and mechanisms of formation that will assist in the development of selection strategies to improve eggshell quality and food safety of the table egg.

Project description:BackgroundEggshell 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.ResultsThe 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.ConclusionsOvocleidin-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.

Project description:This study evaluated the changes in eggshell mechanical properties, ultrastructure, calcium metabolism-related serum indices, and gene expression in eggshell gland during eggshell formation between laying ducks in the peak (young duck) and late phase (aged duck) of production. A total of 84 healthy young (31 wk of age) and 84 healthy aged (65 wk of age) Longyan laying ducks were each divided into 6 replicates of 14 birds, and caged individually. All the ducks were fed in one house with the same corn-soybean meal-based diet for 5 wk. The eggshell mechanical properties (shell proportion, thickness, breaking strength, and fracture toughness) and chemical components (matrix proteins, calcium, phosphorus, and magnesium) decreased in aged laying ducks (P < 0.05). Shell structural indices: total thickness, effective thickness and its proportion decreased, whereas mammillary knob width and its proportion increased (P < 0.05). The regulation values of early fusion, cuffing, caps, and total score of mammillary knobs were higher in aged laying ducks relative to the young ducks (P < 0.05). During the initial, growth and terminal stages of eggshell formation, shell thickness and breaking strength (terminal), shell weight, and its proportion (terminal) decreased in aged laying ducks (P < 0.05). Ultrastructural changes during shell formation indicated that the mammillary-knob density and effective thickness decreased (P < 0.05). Decreases occurred in serum content of phosphorus (growth), and estradiol and calcium contents (terminal) (P < 0.05). Relative expression of Ca2+ transporter and HCO3- exchanger, and matrix proteins genes decreased in aged laying ducks (P < 0.05) at all stages of eggshell formation. Collectively, the decreased incidence of early fusion and caps, increased thickness and width of mammillary knobs, and decreased effective thickness are the crucial differences leading to the compromised mechanical properties of eggshell in the late laying period. A disturbed regulation of calcium metabolism and uterine expression of ion transporters, especially for HCO3- exchange of aged laying ducks likely contribute to age-induced ultrastructural deterioration of the eggshell.

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

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

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