Eggshell Biomineralization Research Articles

Multiscale Mineralization in the Leopard Gecko Eggshell

AbstractReptilian (non‐avian) eggshell biomineralization is markedly different from avian eggshells. Despite discontinuous/variable mineralization, the reptilian eggshell (like in birds) is responsible for protective and developmental functions of the egg as part of a highly successful reproduction strategy. Using multiscale imaging and characterization methods including X‐ray and electron microscopy/tomography, a 3D overview of eggshell ultrastructure in the gekkotan Eublepharis macularius – the common leopard gecko is provided. Eggshell mineral is shown to be sensitive to aqueous preparations, where cryogenic methods are essential for accurate mineral and organic preservation. Interwoven with the mineral are underlying organic fibers involved in the formation of complex hierarchical order in the eggshell, including nucleation and growth of mineral. The calcitic mineral phase of the eggshell takes on three microstructural motifs at the shell surface: nodular scutes (≈40 µm), intermittent blunt or star‐shaped mineral rosettes (≈1 mm), and smaller mineral corollas (≈10 µm). Individual scutes have two hierarchical mineral structures – mineral nanospheres (≈500 nm) and mineral nanodomains (≈40 nm). Diverse and variable biomineralization features of reptilian eggshells likely reflect the importance of differences in ecology and physiology on egg formation, survival, and eggshell mechanics, and are likely related to hydration sensitivity, structural organic‐inorganic interactions, and mineral polymorphism.

Aging-associated increased nitric oxide production is a potential cause of inferior eggshell quality produced by aged laying hens

It is important to prolong the productive life of laying hens without compromising their welfare. Therefore, in this study, we aimed to identify the cause for inferior quality egg production of aged hens by investigating the aging-associated molecular changes related to eggshell formation in the isthmic and uterine mucosae and determining whether nitric oxide plays a role in decreasing the quality of eggs produced by aged hens. Young (35 weeks old) and aged (130 weeks old) White Leghorn laying hens were used in this study to determine the effects of age on the expression of proteins related to eggshell membranes formation in the isthmus and eggshell biomineralization and nitric oxide production in the uterus. Nitric oxide synthesis during the ovulatory cycle was examined in twenty-five laying hens (46–52 weeks old) euthanized at 0, 4, 7, 16, and 24 h after oviposition. S-Nitroso-N-acetylpenicillamine (a nitric oxide donor) was added to the cultured isthmic and uterine mucosal cells to examine the effects of nitric oxide on the expression of genes related to eggshell membranes formation and eggshell biomineralization, respectively. The results showed that the protein abundance of collagen I and V in the isthmic mucosa and collagen V in the eggshell membranes were lower in aged hens than in young hens. The mRNA expression levels of calbindin, osteopontin, and ovocalyxin-36 and the protein abundance of calbindin and carbonic anhydrase-2 were lower in the uterine mucosa of aged hens than in that of young hens. Nitric oxide synthesis was higher in the uterine mucosa of aged hens than in that of young hens. Nitric oxide downregulated the mRNA expression levels of osteopontin and ovocalyxin-36 in cultured uterine mucosal cells. Our results indicated that the eggshell quality decreases with aging due to molecular changes in the uterine mucosa affecting the eggshell membrane formation and eggshell biomineralization. Moreover, nitric oxide overproduction may play a role in this dysfunction.

Integrated Information on the Structure and Composition of the Ostrich Eggshell (Struthio camelus)

Ostrich eggshells are excellent examples of avian biomineralization. Interest in these eggshells is focused on their potential as a food source, example of a biomaterial for medical and industrial applications, and the use of fossil remains for paleoenvironmental reconstructions. Due to this interest, there is some information about aspects of eggshell biomineralization, but it is scattered in different publications and is limited in scope about mineralogy-crystallography and/or composition. Here, we re-examine the biomineralization of the Struthio eggshells focusing on the structure, from macro- to nano-scales, crystallography, and composition of mineral and organic phases. Our results show that there is a very tight biomineralization control, from well-defined structures at nanoscale to precise crystallographic orientation of calcite crystals, in the formation of a biomineral that is unparalleled in other avian eggshells. Overall, this finding would explain the thickness and excellent mechanical properties of ostrich eggshells.

Antimicrobial Proteins and Peptides in Avian Eggshell: Structural Diversity and Potential Roles in Biomineralization.

The calcitic avian eggshell provides physical protection for the embryo during its development, but also regulates water and gaseous exchange, and is a calcium source for bone mineralization. The calcified eggshell has been extensively investigated in the chicken. It is characterized by an inventory of more than 900 matrix proteins. In addition to proteins involved in shell mineralization and regulation of its microstructure, the shell also contains numerous antimicrobial proteins and peptides (AMPPs) including lectin-like proteins, Bacterial Permeability Increasing/Lipopolysaccharide Binding Protein/PLUNC family proteins, defensins, antiproteases, and chelators, which contribute to the innate immune protection of the egg. In parallel, some of these proteins are thought to be crucial determinants of the eggshell texture and its resulting mechanical properties. During the progressive solubilization of the inner mineralized eggshell during embryonic development (to provide calcium to the embryo), some antimicrobials may be released simultaneously to reinforce egg defense and protect the egg from contamination by external pathogens, through a weakened eggshell. This review provides a comprehensive overview of the diversity of avian eggshell AMPPs, their three-dimensional structures and their mechanism of antimicrobial activity. The published chicken eggshell proteome databases are integrated for a comprehensive inventory of its AMPPs. Their biochemical features, potential dual function as antimicrobials and as regulators of eggshell biomineralization, and their phylogenetic evolution will be described and discussed with regard to their three-dimensional structural characteristics. Finally, the repertoire of chicken eggshell AMPPs are compared to orthologs identified in other avian and non-avian eggshells. This approach sheds light on the similarities and differences exhibited by AMPPs, depending on bird species, and leads to a better understanding of their sequential or dual role in biomineralization and innate immunity.

Myostatin Mutation in Japanese Quail Increased Egg Size but Reduced Eggshell Thickness and Strength.

Simple SummaryAvian eggs provide huge benefits to both science and society by providing an important model for developmental studies and a high-quality protein source for the human diet. Especially, the hard-shell layer existing at the outer part of eggs is a unique characteristic, which is exclusive in avian species compared to other egg-laying species. Among various avian models developed to investigate genetic factors for potential industrial application, myostatin (MSTN) mutations in quail and chickens were recently generated, resulting in improved meat yield. In addition to previously reported growth and egg production traits in MSTN mutant quail, eggshell quality of mutants was further investigated in this study. Although eggshell height, width, and weight were increased by the MSTN mutation, eggshell breaking strength (EBS) and eggshell thickness were decreased in mutant eggs compared to wild-type eggs. Although these data indicated that decreased eggshell thickness contributed to decreased EBS in mutant eggs, the cellular mechanism of thinner eggshell formation in uterus by MSTN mutation needs to be further investigated using MSTN mutant quail.Recently developed myostatin (MSTN) mutant quail and chickens demonstrated similar effects of MSTN on muscle and fat developments between avian and mammalian species. However, the effect of MSTN mutation on the quality of eggshells, an important avian specific characteristic, has not yet been investigated although egg production traits of mutant quail have been studied. In this study, several parameters for eggshell quality, including eggshell size, eggshell weight, eggshell breaking strength (EBS), and eggshell thickness, were all compared between MSTN mutant and wild-type (WT) eggs. MSTN mutant eggs had greater height and width along with heavier eggshell weight compared to WT eggs, which shows proportional improvement in egg size as affected by the MSTN mutation. However, EBS and eggshell thickness were decreased in mutant eggs compared to WT eggs. In addition, the palisade layer, the thickest and most important layer for the strength of an eggshell, was also decreased without a change in the number of vesicular holes. These data indicated that decreases in the thickness of the eggshell and the palisade layer would be a main factor contributing to a lower EBS in mutant eggs. MSTN mutant quail provide a useful model to better understand the function of MSTN on avian uterine cell development and eggshell biomineralization.

A transcriptome analysis for 24-hour continuous sampled uterus reveals circadian regulation of the key pathways involved in eggshell formation of chicken

Circadian timing system controlled the rhythmic events, for example, ovulation and oviposition in chickens. However, how biological clock mediates eggshell formation remains obscure. Here, A 24-h mRNA transcriptome analysis was carried out in the uterus of 18 chickens with similar oviposition time points to identify the rhythmic genes and to reveal critical genes and biological pathways involved in the eggshell biomineralization. JTK_CYCLE analysis and real-time PCR revealed a total of 1,793 genes from the sequencing database with 23,513 genes (FPKM>1) were rhythmic genes regulating the rhythmic system and the expression of typical clock genes Per2, Cry1, Bmal1, Clock, Per3, and Rev-erbβ were rhythmically expressed, which suggested that endogenous clock in uterus might control the eggshell mineralization. Time of peak expression of the rhythmic genes was analyzed based on their acrophase. The main phases clustered at the periods from Zeitgeber time 0 (ZT0) to ZT4 (6:00–10:00) and from ZT10 to ZT14 (16:00-20:00). The rhythmic genes were annotated to the following Gene Ontology terms rhythmic process, lyase, ATP binding, cell membrane component. KEGG pathway enrichment analysis revealed the top 15 rhythmic genes were involved in vital biological pathways, including syndecan (1, 2, 3)-mediated signaling, post-translational regulation of adheres junction stability and disassembly, FoxO family signaling, TGF-β receptor and transport of small molecular pathways. 166 of total 1,235 genes (13.4%) were defined as rhythmic transfer factors (TFs) and they were investigated expression time distribution of cis-elements of circadian clock system D-box, E-box, B-site, and Y-Box within 24 h. Results indicated that rhythmic TFs at each phase are potential drivers of their circadian transcription activities. Compared with the control, the expression abundances of ion transport elements SCNN1G, CA2, SPP1, and ATP1B1 were significantly decreased after the interference of Bmal1 gene in synchronized uterine tubular gland cells. Clock genes changed their expression along with the eggshell formation, indicating that there is circadian clock in the uterus of chicken and it regulates the expression of eggshell formation genes.

Three chromosome-level duck genome assemblies provide insights into genomic variation during domestication

Domestic ducks are raised for meat, eggs and feather down, and almost all varieties are descended from the Mallard (Anas platyrhynchos). Here, we report chromosome-level high-quality genome assemblies for meat and laying duck breeds, and the Mallard. Our new genomic databases contain annotations for thousands of new protein-coding genes and recover a major percentage of the presumed “missing genes” in birds. We obtain the entire genomic sequences for the C-type lectin (CTL) family members that regulate eggshell biomineralization. Our population and comparative genomics analyses provide more than 36 million sequence variants between duck populations. Furthermore, a mutant cell line allows confirmation of the predicted anti-adipogenic function of NR2F2 in the duck, and uncovered mutations specific to Pekin duck that potentially affect adipose deposition. Our study provides insights into avian evolution and the genetics of oviparity, and will be a rich resource for the future genetic improvement of commercial traits in the duck.

Effect of organic and inorganic dietary selenium supplementation on gene expression in oviduct tissues and Selenoproteins gene expression in Lohman Brown-classic laying hens

BackgroundThe oviduct of a hen provides a conducive environment for egg formation, which needs a large amount of mineral elements from the blood via trans-epithelial permeability. Eggshell is the calcified layer on the outside of an egg that provides protection and is critical for egg quality. However, little is known about the genes or proteins involved in eggshell formation, and their relationship to dietary microminerals. We hypothesized that dietary selenium supplementation in chickens will influence genes involved in eggshell biomineralization, and improve laying hen antioxidant capacity. The objective of this research was to investigate how organic and inorganic dietary selenium supplementation affected mRNA expression of shell gland genes involved in eggshell biomineralization, and selenoproteins gene expression in Lohman Brown-Classic laying hens.ResultsShell gland (Uterus) and liver tissue samples were collected from hens during the active growth phase of calcification (15–20 h post-ovulation) for RT-PCR analysis. In the oviduct (shell gland and magnum) and liver of laying hens, the relative expression of functional eggshell and hepatic selenoproteins genes was investigated. Results of qPCR confirmed the higher (p < 0.05) mRNA expression of OC-17 and OC-116 in shell gland of organic Se hen compared to inorganic and basal diet treatments. Similarly, dietary Se treatments affected the mRNA expression of OCX-32 and OCX-36 in the shell gland of laying hens. In the magnum, mRNA expression of OC-17 was significantly (p < 0.05) higher in hens fed-bacterial organic, while OC-116 mRNA expression was down-regulated in dietary Se supplemented groups compared to non-Se supplemented hens. Moreover, when compared to sodium selenite, only ADS18 bacterial Se showed significantly (p < 0.05) higher mRNA levels in GPX1, GPX4, DIO1, DIO2 and SELW1, while Se-yeast showed significantly (p < 0.05) higher mRNA levels in TXNRD1 than the non-Se group.ConclusionsDietary Se supplementation especially that from a bacterial organic source, improved shell gland and hepatic selenoproteins gene expression in laying hens, indicating that it could be used as a viable alternative source of Se in laying hens. The findings could suggest that organic Se upregulation of shell gland genes and hepatic selenoproteins in laying hens is efficient.

Evolution of the Avian Eggshell Biomineralization Protein Toolkit - New Insights From Multi-Omics.

The avian eggshell is a remarkable biomineral, which is essential for avian reproduction; its properties permit embryonic development in the desiccating terrestrial environment, and moreover, are critically important to preserve unfertilized egg quality for human consumption. This calcium carbonate (CaCO3) bioceramic is made of 95% calcite and 3.5% organic matrix; it protects the egg contents against microbial penetration and mechanical damage, allows gaseous exchange, and provides calcium for development of the embryonic skeleton. In vertebrates, eggshell occurs in the Sauropsida and in a lesser extent in Mammalia taxa; avian eggshell calcification is one of the fastest known CaCO3 biomineralization processes, and results in a material with excellent mechanical properties. Thus, its study has triggered a strong interest from the researcher community. The investigation of eggshell biomineralization in birds over the past decades has led to detailed characterization of its protein and mineral constituents. Recently, our understanding of this process has been significantly improved using high-throughput technologies (i.e., proteomics, transcriptomics, genomics, and bioinformatics). Presently, more or less complete eggshell proteomes are available for nine birds, and therefore, key proteins that comprise the eggshell biomineralization toolkit are beginning to be identified. In this article, we review current knowledge on organic matrix components from calcified eggshell. We use these data to analyze the evolution of selected matrix proteins and underline their role in the biological toolkit required for eggshell calcification in avian species. Amongst the panel of eggshell-associated proteins, key functional domains are present such as calcium-binding, vesicle-binding and protein-binding. These technical advances, combined with progress in mineral ultrastructure analyses, have opened the way for new hypotheses of mineral nucleation and crystal growth in formation of the avian eggshell, including transfer of amorphous CaCO3 in vesicles from uterine cells to the eggshell mineralization site. The enrichment of multi-omics datasets for bird species is critical to understand the evolutionary context for development of CaCO3 biomineralization in metazoans, leading to the acquisition of the robust eggshell in birds (and formerly dinosaurs).

Expression of follistatin is associated with egg formation in the oviduct of laying hens.

The objective of this study was to examine the expression profiles of follistatin (FST) and its associated molecules (MSTN, INHA, INHBB, INHBA, ACVR2A, and ACVR2B) in the oviduct of laying hens at 3hr and 20hr post-ovulation (p.o., n=5; 35weeks old), molting (n=5; 60weeks old), and non-laying (n=4; 35-60weeks old) hens and also to localize the FST by using immunohistochemistry assay. Expression of FST was significantly higher (p<.05), and MSTN was lower in the uterus of laying hens around 15-20hr p.o. (during eggshell formation), however, their expressions in the magnum remain unchanged across different physiological stages of hens. FST was mainly expressed in the luminal and glandular epithelium of the uterine tissues, and their expression intensity was highest in laying hens during the eggshell mineralization. There was a relatively increased expression of INHA in the magnum of laying hens around 3hr p.o. as compared to non-laying and molting hens. At the same time (3hr p.o.), there was a significant (p<.05) decrease in the expression of the INHBB, ACVR2A, and ACV2B. These results indicate that follistatin may regulate the differentiation of uterine luminal and glandular epithelium during eggshell biomineralization.

Candidate genes of the transcellular and paracellular calcium absorption pathways in the small intestine of laying hens

ABSTRACTTo meet the high calcium (Ca) demand during eggshell biomineralization (2 g of Ca per egg), laying hens develop specific metabolic regulations to maintain Ca homeostasis. The intake of Ca, its solubilization, and absorption capacity are enhanced at sexual maturity (SM). A better knowledge of the intestinal Ca transporters involved in their variations at this stage could indicate new nutritional strategies to enhance Ca digestive utilization. Transcellular Ca absorption pathway and its major player calbindin-D 28 K (CALB1) mediate a saturable transport, which has been extensively described in this model. Conversely, a contribution by the paracellular pathway involving non-saturable Ca transport through intercellular tight junction has also been suggested. The aim of the present study was to identify candidate genes of these two pathways and their patterns of expression, in immature pullets (12, 15, and 17 wk old) and mature laying hens (23 wk old) in the duodenum, jejunum, and ileum. Using RT-qPCR, this study identifies 3 new candidate genes for transcellular, and 9 for paracellular Ca transport. A total of 5 candidates of the transcellular pathway, transient receptor potential cation channels subfamily C member 1 (TRPC1) and M member 7 (TRPM7); CALB1 and ATPase plasma membrane Ca2+ transporting 1 (ATP2B1) and ATPase plasma membrane Ca2+ transporting 2 (ATP2B2) were enhanced with age or after SM in the duodenum, the jejunum or all 3 segments. A total of 4 candidates of the paracellular pathway Claudin 2 (CLDN2) and tight junction proteins 1, 2, and 3 (TJP1, TJP2 and TJP3) increased in the small intestine after SM. Additionally, CALB1, ATP2B2, and CLDN2 were overexpressed in the duodenum or the jejunum or both segments after SM. The enhanced expression of candidate genes of the paracellular Ca pathway after SM, supports that the non-saturable transport could be a mechanism of great importance when high concentrations of soluble Ca are observed in the intestinal content during eggshell formation. Both pathways may work cooperatively in the duodenum and jejunum, the main sites of Ca absorption in laying hens.

RNA sequencing-based analysis of the laying hen uterus revealed the novel genes and biological pathways involved in the eggshell biomineralization

Eggshell is the outermost calcified covering of an egg that protects it from microbial invasion and physical damage, and is critical for egg quality. However, understanding of the genes/proteins and the biological pathways regulating the eggshell formation is still obscure. We hypothesized that the transcriptomic analysis of the chicken uteri using RNA-sequencing may reveal novel genes and biological pathways involved in the eggshell biomineralization. RNA-sequence analysis using uteri of laying hens at 15–20 h post-ovulation (layers, n = 3) and non-laying (non-layers, n = 3) hens was carried out. About 229 differentially expressed genes (DEGs) were up-regulated in the layers compared to the non-layers. Kyoto Encyclopedia of Genes and Genomes (KEGG) and Ingenuity Pathway Analysis (IPA) revealed more than ten novel genes and biological pathways related to calcium transport and mineralization in the uterus. Based on the enriched pathways and molecular function analysis, 12 DEGs related to eggshell mineralization were further analyzed in the uteri of layers (3 h and 15–20 h post-ovulation), non-layers and molters using qPCR. Expressions of OC-116 (regulator of mineralization), OTOP2 (modulator of cellular calcium influx), CALCB (intracellular release of Ca-ions), STC2 (increases alkaline phosphatase activity), and ATP2C2 (cellular import of Ca-ions) were significantly higher in the uteri of laying hen at 15–20 h post-ovulation. This study identified the involvement of novel genes and their proposed biological pathways in the regulation of eggshell formation.

Expression of pro- and anti-inflammatory cytokines and chemokines during the ovulatory cycle and effects of aging on their expression in the uterine mucosa of laying hens

The aim of this study was to examine whether cytokines and chemokines expressed in the uterine mucosa play a role in the process of eggshell formation in the chicken uterus. Changes in the expression levels of pro- and anti-inflammatory cytokines and chemokines in the uterine mucosa during an ovulatory cycle (experiment 1) and effects of aging on their expression (experiment 2) were examined. In experiment 1, the expression of the pro-inflammatory cytokines IL1β, IL6, TNFSF15, and IFNγ, and a chemokine CX3CL1 was found to increase during eggshell biomineralization (16 h following oviposition), while anti-inflammatory TGFβ2 expression was found to increase at 4 h following oviposition. In experiment 2, a higher expression of the anti-inflammatory cytokines TGFβ2 and TGFβ3, and chemokines CXCLi2 and CX3CL1, was observed in aged hens than in young hens. A significantly higher number of macrophages and CD8+ T cells were observed in the uterine tissue of aged hens than in young hens. Furthermore, the expression of adhesion molecules associated with leukocytic infiltration was found to be higher in aged hens than in young hens. We conclude that the eggshell formation process may be affected by the pro- and anti-inflammatory cytokines and chemokines. The balanced expressions of these molecules might be disrupted in aged hens.

Comparison of the structure, crystallography and composition of eggshells of the guinea fowl and graylag goose

The structure and composition of the eggshells of two commercial species (guinea fowl and greylag goose) have been studied. Thin sections and scanning electron microcopy show the similarity of the overall structure, but the relative thickness of the layers differs in these two taxa. Atomic force microscopy shows that the different layers are composed of rounded, heterogeneous granules, the diameter of which is between 50 and 100nm, with a thin cortex. Infrared data and thermogravimetric analyses show that both eggshells are made of calcite, but differing on the quality and quantity when the organic component is considered. Chemical maps show that chemical element distribution is not uniform within a sample, and differs between the species, but with low magnesium content. Electron back scattered diffraction confirms the eggshells are calcite, but the microtexture strongly differs between the two species. Based on the chemical-structural differences, a species-specific biological control on the biomineralization is found, despite the rapid formation of an eggshell. Overall results indicate that to estimate the quality of eggshells, such as resistance to breakage, is not a straightforward process because of the high complexity of avian eggshell biomineralization.

Data set for the proteomic inventory and quantitative analysis of chicken uterine fluid during eggshell biomineralization.

Chicken eggshell is the protective barrier of the egg. It is a biomineral composed of 95% calcium carbonate on calcitic form and 3.5% organic matrix proteins. Mineralization process occurs in uterus into the uterine fluid. This acellular fluid contains ions and organic matrix proteins precursors which are interacting with the mineral phase and control crystal growth, eggshell structure and mechanical properties. We performed a proteomic approach and identified 308 uterine fluid proteins. Gene Ontology terms enrichments were determined to investigate their potential functions. Mass spectrometry analyses were also combined to label free quantitative analysis to determine the relative abundance of 96 proteins at initiation, rapid growth phase and termination of shell calcification. Sixty four showed differential abundance according to the mineralization stage. Their potential functions have been annotated. The complete proteomic, bioinformatic and functional analyses are reported in Marie et al., J. Proteomics (2015) [1].

Quantitative proteomics and bioinformatic analysis provide new insight into protein function during avian eggshell biomineralization.

The calcitic avian eggshell protects the developing embryo and, moreover, ensures that the nutritious table egg remains free of pathogens. The eggshell is formed by nucleation upon a fibrous scaffold (the eggshell membranes) followed by an interaction between the growing mineral crystals and the shell organic matrix. This interaction leads to a highly ordered shell microstructure and texture which contribute to its exceptional mechanical properties. Shell mineralization occurs in three distinct phases of calcification (initiation, growth and termination), which are associated with distinct populations of matrix proteins that are secreted into the acellular uterine fluid as modulators of the process. The recent development of high-throughput methods has led to the identification of many proteins in the shell, but little is known concerning their role in shell formation. In order to determine precisely the importance of particular proteins relative to eggshell mineralization, this project used qualitative and quantitative proteomics of the uterine fluid constituents, coupled with bioinformatic analysis, to predict the functional role of proteins secreted at each of the three main stages of shell calcification. Besides its relevance to food production and to hen reproduction, eggshell calcification is furthermore a relevant model for studying calcium carbonate biomineralization on a two-dimensional membrane support. Better understanding of this process will provide insight into the fabrication of ceramics at ambient pressure and temperature.

Integrating De Novo Transcriptome Assembly and Cloning to Obtain Chicken Ovocleidin-17 Full-Length cDNA

Efficiently obtaining full-length cDNA for a target gene is the key step for functional studies and probing genetic variations. However, almost all sequenced domestic animal genomes are not ‘finished’. Many functionally important genes are located in these gapped regions. It can be difficult to obtain full-length cDNA for which only partial amino acid/EST sequences exist. In this study we report a general pipeline to obtain full-length cDNA, and illustrate this approach for one important gene (Ovocleidin-17, OC-17) that is associated with chicken eggshell biomineralization. Chicken OC-17 is one of the best candidates to control and regulate the deposition of calcium carbonate in the calcified eggshell layer. OC-17 protein has been purified, sequenced, and has had its three-dimensional structure solved. However, researchers still cannot conduct OC-17 mRNA related studies because the mRNA sequence is unknown and the gene is absent from the current chicken genome. We used RNA-Seq to obtain the entire transcriptome of the adult hen uterus, and then conducted de novo transcriptome assembling with bioinformatics analysis to obtain candidate OC-17 transcripts. Based on this sequence, we used RACE and PCR cloning methods to successfully obtain the full-length OC-17 cDNA. Temporal and spatial OC-17 mRNA expression analyses were also performed to demonstrate that OC-17 is predominantly expressed in the adult hen uterus during the laying cycle and barely at immature developmental stages. Differential uterine expression of OC-17 was observed in hens laying eggs with weak versus strong eggshell, confirming its important role in the regulation of eggshell mineralization and providing a new tool for genetic selection for eggshell quality parameters. This study is the first one to report the full-length OC-17 cDNA sequence, and builds a foundation for OC-17 mRNA related studies. We provide a general method for biologists experiencing difficulty in obtaining candidate gene full-length cDNA sequences.

Structure, Self-Assembly, and Dual Role of a β-Defensin-like Peptide from the Chinese Soft-Shelled Turtle Eggshell Matrix

Biomineral matrix formation and molecular recognition are two important processes associated with eggshell biomineralization. To understand these two processes, a major intracrystalline peptide, pelovaterin, was isolated from turtle (Pelodiscus sinensis) eggshell and its tertiary and quaternary structures were established. The global fold of pelovaterin is similar to that of human beta-defensins but has a large hydrophobic core and a short hydrophilic N-terminal segment, which is not preserved in defensins. Pelovaterin exhibits strong antimicrobial activity against two pathogenic gram-negative bacteria, Pseudomonas aeruginosa and Proteus vulgaris, and stabilizes a thin film of metastable vaterite. We show that pelovaterin self-aggregates in the form of micellar nanospheres and the aggregation in solution is entropy-driven. It is suggested that the micellar aggregation of pelovaterin is responsible for the induction and stabilization of the metastable phase by altering the interfacial energy. The results demonstrate the adaptability of an extracellular matrix protein to perform multiple tasks: polymorph discrimination and protection of the contents of the egg against bacterial invasion.

Formation of Transient Amorphous Calcium Carbonate Precursor in Quail Eggshell Mineralization: An In Vitro Study

To understand the mechanism of quail eggshell biomineralization, we have performed two CaCO(3) precipitation experiments. In the reprecipitation experiments, supersaturated Ca(HCO(3))(2) was prepared by bubbling CO(2) through a slurry of biogenic CaCO(3) obtained from bleach-treated eggshell followed by filtration to obtain a clear solution for crystallization experiments. The nucleated crystals were collected at various time intervals and analyzed. In the second experiment, the extracted SOM from the bleach-treated eggshell was added to the supersaturated clear solution of Ca(HCO(3))(2) solution obtained by bubbling CO(2) gas through a slurry of synthetic CaCO(3) followed by filtration. The crystals/precipitates collected at various time intervals were analyzed. Both experiments showed that amorphous CaCO(3) (ACC) was precipitated in the early stages, which then transformed to the most stable crystalline calcite phase. Amino acid analysis of the soluble organic matrixes (SOM) indicated the presence of high amounts of Glx and Asx amino acids. Ovomucoid--an acidic glycoprotein, and lysozyme--a basic protein, are the two major components along with a few low molecular weight peptides present in the SOM of quail eggshell matrix. Both ovomucoid and lysozyme did not induce precipitation of the ACC phase in in vitro conditions, while the fraction containing low molecular weight peptides induced the precipitation of ACC, suggesting that the latter play an important role in the eggshell biomineralization. Thus, organisms can produce inorganic minerals which assume nonequilibrium morphologies and intricate architecture by precipitating transient ACC, which then transformed into the crystalline phase. Altogether, these observations further demonstrate that this strategy may be common in both vertebrate and invertebrate mineralized structures.

Crystal growth and the role of the organic network in eggshell biomineralization

A model based on geometrical crystal growth considerations is proposed for the deposition of the crocodilian, testudinian and avian eggshells. Ir each shell column, crystal deposition is initiated at a single location, from which growth fans out at all angles to the shell normal. In both co1citic and aragonitic shells, growth is in the [001] direction, resulting in an increase in the degree of (001) preferred orientation with distance from nucleation. Where there is unhindered crystal growth, the shells show a crystalline fracture morphology, and the degree of texture that develops is a simple function of the column radius. This type of growth makes up the whole of the testudinian shell, the inner 0.3-0.4 (30-40 %) of the thick ratite shells and the cone layer of the other avian shells. At the start of the palisade layer of the avian shell, the onset of deposition of the organic component coincides with a hindrance to texture development, which thereafter proceeds at a lower rate. A further hindrance occurs about halfway through the shell, probably caused by a change in the physical characteristics of the organic network. The degree of texture that develops in the avian shell is a function of the column radius and the degree of physical hindrance presented by the organic network. The palisade layer of the avian shell has a composite fracture morphology resulting from the intermingling of the network with the inorganic phase.The organic component does not appear to control crystal growth, as previously believed, but instead acts as a reinforcing fibrous network.