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Epigenetic information is transmitted from cell to cell, and even generation to generation. The question of epigenetic inheritance in fish has become of crucial interest in the recent years, when the mammalian model of methylome erasure in germ cells and embryos was found not to be conserved. Fish, which are particularly exposed to environmental variations might thus be prone to transmit epigenetic alterations to their offspring, driving rapid environmental acclimation. Here, by sequencing spermatozoa and muscle methylomes, we characterized the methylation landscape of paternal gametes in rainbow trout and demonstrated its sensitivity to a 4°C increased rearing temperature during spermatogenesis. We found that spermatozoa methylome primes housekeeping and developmental genes for activation and might be instrumental to early development. Most of these methylation-free promoters were not affected by temperature, attesting the robustness of the epigenetic programming of early development. However, the increase of temperature triggered the differential methylation of 5,359 regions, among which 560 gene promoters control spermiogenesis and lipid metabolism. We therefore report, for the first time in fish, that sperm epigenetic landscape carries marks of parental environmental conditions. In the context of a 4°C temperature increase during spermatogenesis, we describe how rainbow trout sperm DNA methylation might be a molecular basis of intergenerational inheritance and question its role in controlling next generation’s performances and acclimation to climate change.

BackgroundThe thermal-manipulation (TM) during egg incubation is a cyclic exposure to hot or cold temperatures during embryogenesis that is associated to long-lasting effects on growth performance, physiology, metabolism and temperature tolerance in birds. An increase of the incubation temperature of Japanese quail eggs affected the embryonic and post-hatch survival, growth, surface temperatures and blood characteristics potentially related to thermoregulation capacities. To gain new insights in the molecular basis of TM in quails, we investigated by RNA-seq the hypothalamus transcriptome of 35 days-old male and female quails that were treated by TM or not (C, control) during embryogenesis and that were exposed (HC) or not (RT) to a 36 °C heat challenge for 7 h before sampling.ResultsFor males, 76, 27, 47 and 0 genes were differentially expressed in the CHC vs. CRT, CRT vs. TMRT, TMHC vs. TMRT and CHC vs. TMHC comparisons, respectively. For females, 17, 0, 342 and 1 genes were differentially expressed within the same respective comparisons. Inter-individual variability of gene expression response was observed particularly when comparing RT and HC female animals. The differential expression of several genes was corroborated by RT-qPCR analysis. Gene Ontology functional analysis of the differentially expressed genes showed a prevalent enrichment of terms related to cellular responses to stimuli and gene expression regulation in both sexes. Gene Ontology terms related to the membrane transport, the endoplasmic reticulum and mitochondrial functions as well as DNA metabolism and repair were also identified in specific comparisons and sexes.ConclusionsTM had little to no effect on the regulation of gene expression in the hypothalamus of 35 days-old Japanese quails. However, the consequences of TM on gene expression were revealed by the HC, with sex-specific and common functions altered. The effects of the HC on gene expression were most prominent in TM females with a ~ 20-fold increase of the number of differentially expressed genes, suggesting that TM may enhance the gene response during challenging conditions in female quail hypothalamus. TM may also promote new cellular strategies in females to help coping to the adverse conditions as illustrated by the identification of differentially expressed genes related to the mitochondrial and heat-response functions.

The broiler industry is facing an increasing prevalence of breast myopathies, such as white striping (WS) and wooden breast (WB), and the precise aetiology of these occurrences remains poorly understood. To progress our understanding of the structural changes and molecular pathways involved in these myopathies, a transcriptomic analysis was performed using an 8 × 60 K Agilent chicken microarray and histological study. The study used pectoralis major muscles from three groups: slow-growing animals (n = 8), fast-growing animals visually free from defects (n = 8), or severely affected by both WS and WB (n = 8). In addition, a weighted correlation network analysis was performed to investigate the relationship between modules of co-expressed genes and histological traits. Functional analysis suggested that selection for fast growing and breast meat yield has progressively led to conditions favouring metabolic shifts towards alternative catabolic pathways to produce energy, leading to an adaptive response to oxidative stress and the first signs of inflammatory, regeneration and fibrosis processes. All these processes are intensified in muscles affected by severe myopathies, in which new mechanisms related to cellular defences and remodelling seem also activated. Furthermore, our study opens new perspectives for myopathy diagnosis by highlighting fine histological phenotypes and genes whose expression was strongly correlated with defects.

Background: In mammals, oxidative stress (OS) induces negative effects on reproductive functions. In broiler hens, the genetic selection has increased susceptibility to OS. Grape seed extracts (GSE) are known as potent antioxidants. Here, we investigated the effects of time and level of GSE dietary supplementation on egg production and quality, fertility parameters, Reactive Oxygen Species (ROS) and steroid content in yolk egg associated to plasma adipokines in broiler hens. Methods: We designed an in vivo study using four groups of hens: A (control), B and C (supplemented with GSE at 0.5% and 1% of the total diet composition, respectively, starting at week 4) and D (supplemented with GSE at 1% of the total diet composition since hatching). We assessed the egg production from 23 th to 40 th weeks and quality at 33 th week. After artificial inseminations, the fertility parameters were calculated. In egg yolk, Reactive Oxygen Species (ROS) level and steroid production were evaluated by Ros-Glo H202 and ELISA assay, respectively. Expression of steroidogenic enzymes and adipokines and their receptors was determined by RT-qPCR in ovarian cells and plasma adipokines (RARRES2, ADIPOQ and NAMPT) were evaluated by specific ELISA assays. Results: The fertility parameters and egg production were unaffected by GSE supplementation. However, the rate of double-yolk eggs decreased for all GSE supplemented. Eggs from the C group were bigger and larger. The shell elasticity was higher for B and C groups. In the yolk egg, GSE supplementation reduced ROS content and steroidogenesis consistent with a decrease in P450 aromatase and StAR mRNA expression and basal in vitro progesterone secretion in granulosa cells. Interestingly, RARRES2 plasma levels were positively correlated while ADIPOQ and NAMPT plasma levels were negatively correlated, with steroids and ROS in yolk. Conclusions: Maternal dietary GSE supplementation did not affect egg production and fertility parameters whereas it reduced ROS content and steroidogenesis in yolk egg. Furthermore, it ameliorated egg quality by decreasing the number of double-yolk eggs and by improving the size of normal eggs and the elasticity of the shell. Taken together, our data suggest the possibility of using dietary maternal GSE to improve egg quality.

International audience; Uterine fluid is an aqueous milieu to which sperm are exposed during their storage and ascent. In this study, a bottom-up proteomic strategy and bioinformatic analysis of hen uterine fluid was performed to improve the understanding of this fluid and its potential role in sperm survival mechanisms. The proteomic data were submitted to ProteomeXchange. Among the 913 proteins identified, 160 are known to be secreted and 640 are referenced in exosomes databases. We isolated exosomes from the avian uterine fluid, analyzed them using electron microscopy, and targeted several exosomes markers (ANXA1/2/4/5, VCP, HSP90A, HSPA8, PARK7, and MDH1) using immunoblotting. Electron microscopy and immunohistochemistry were also used to analyze uterovaginal junctions for the exosomal proteins ANXA4, VCP, and PARK7. Exosomes were observed both at the surface epithelium and inside sperm storage tubules. Our data were compared with two previously published studies on proteomic of hen uterine fluid, and with one study describing the proteomic content of rooster seminal plasma and sperm. In conclusion, we demonstrated for the first time that avian uterine fluid contains exosomes. These may play a key role in preserving sperm functions within the female genital tract. Their presence in the sperm storage tubules may represent an important mechanism regarding interaction between the female genital tract and sperm.

The main objective of ViSEAGO package is to carry out a data mining of biological functions and establish links between genes involved in the study. We developed ViSEAGO in R to facilitate functional Gene Ontology (GO) analysis of complex experimental design with multiple comparisons of interest. It allows to study large-scale datasets together and visualize GO profiles to capture biological knowledge. The acronym stands for three major concepts of the analysis: Visualization, Semantic similarity and Enrichment Analysis of Gene Ontology. It provides access to the last current GO annotations, which are retrieved from one of NCBI EntrezGene, Ensembl or Uniprot databases for several species. Using available R packages and novel developments, ViSEAGO extends classical functional GO analysis to focus on functional coherence by aggregating closely related biological themes while studying multiple datasets at once. It provides both a synthetic and detailed view using interactive functionalities respecting the GO graph structure and ensuring functional coherence supplied by semantic similarity. ViSEAGO has been successfully applied on several datasets from different species with a variety of biological questions. Results can be easily shared between bioinformaticians and biologists, enhancing reporting capabilities while maintaining reproducibility. ViSEAGO is publicly available on https://bioconductor.org/packages/ViSEAGO .

International audience; The Guinea fowl eggshell is a bioceramic material with the remarkable mechanical property of being twice as strong as the chicken eggshell. Both eggshells are composed of 95% calcite and 3.5% organic matrix, which control its structural organization. Chicken eggshell is made of columnar calcite crystals arranged vertically. In the Guinea fowl, the same structure is observed in its inner half, followed by a dramatic change in crystal size and orientation in the outer region. Guinea fowl eggshell is thicker than chicken eggshell. Both structure and shell thickness confer a superior resistance to breakage compared to eggshells of other bird species. To understand the underlying mechanisms controlling the structural organization of this highly resistant material, we used quantitative proteomics to analyze the protein composition of the Guinea fowl eggshell organic matrix at key stages of the biomineralization process. We identified 149 proteins, which were compared to other bird eggshell proteomes and analyzed their potential functions. Among the 149 proteins, 9 are unique to Guinea fowl, some are involved in the control of the calcite precipitation (Lysozyme, Ovocleidin-17-like, Ovocleidin-116 and Ovalbumin), 61 are only found in the zone of microstructure shift and 17 are more abundant in this zone. Significance: The avian eggshell is a critical physical barrier to protect the contents of this autonomous reproductive enclosure from physical and microbial assault. The Guinea fowl (Numida meleagris) eggshell exhibits a unique microstructure (texture), which confers exceptional mechanical properties compared to eggshells of other species. In order to understand the mechanisms that regulate formation of this texture in the Guinea fowl eggshell, we performed comparative quantitative proteomics at key stages of shell mineralization and particularly during the dramatic shift in shell microstructure. We demonstrate that the Guinea fowl eggshell proteome comprises 149 proteins, of which 61 were specifically associated with the change in size and orientation of calcite crystals. Comparative proteomics analysis with eggshell of other bird species leads to new insights into the biomineralization process. Moreover, our data represents a list of organic compounds as potential additives to regulate material design for industrial fabrication of ceramics. This information also provides molecular markers for efficient genomic selection of chicken strains to lay eggs with improved shell mechanical properties for enhanced food safety.

In the last 15 years, the development of functional genomics has increased the number of egg proteins identified from 50 to about 1300. These proteins are initially present in eggs to support a harmonious embryonic development. Consequently, this closed embryonic chamber contains molecules exhibiting diverse functions, including defense, nutrition and many predicted biological activities, which have been investigated using both bioinformatics and experimental investigations. In this chapter, we focus on some very interesting activities of high potential reported for minor egg proteins (excluding ovalbumin, ovotransferrin and lysozyme). The shell matrix proteins are involved in the calcification process to define and control the final texture of the shell and thereby its mechanical properties. Antimicrobial proteins are part of innate immunity and are mainly present in the white and vitelline membranes. They encompass several protein families, including protease inhibitors, vitamin-binding proteins, defensins, LBP-PLUNC family proteins and heparin-binding proteins. The egg also possesses additional bioactive proteins with direct anti-cancerous and antioxidant activities or whose biochemical properties are currently used to develop diagnostic tools and strategies for targeted therapy. Finally, this chapter also reports some emerging functions in tissue remodeling/wound healing and proposes some relevant bioactive candidates and research fields that would be interesting to investigate further.

International audience; To 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.

cmi13027-sup-0001-Table_S1.xlsx cmi13027-sup-0002-Table_S2.xlsx cmi13027-sup-0003-Table_S3.docx cmi13027-sup-0004-suppl_figures.pptx; International audience; Coccidia are obligate intracellular protozoan parasites responsible for human and veterinary diseases. Eimeria tenella, the etiologic agent of cecal coccidiosis, is a major pathogen of chickens. In Toxoplasma gondii, some kinases from the rhoptry compartment (ROP) are key virulence factors. ROP kinases hijack and modulate many cellular functions and pathways, allowing T. gondii survival and development. E. tenella's kinome comprises 28 putative members of the ROP kinase family; most of them are predicted, as pseudokinases and their functions have never been characterized. One of the predicted kinase, EtROP1, was identified in the rhoptry proteome of E. tenella sporozoites. Here, we demonstrated that EtROP1 is active, and the N-terminal extension is necessary for its catalytic kinase activity. Ectopic expression of EtROP1 followed by co-immunoprecipitation identified cellular p53 as EtROP1 partner. Further characterization confirmed the interaction and the phosphorylation of p53 by EtROP1. E. tenella infection or overexpression of EtROP1 resulted both in inhibition of host cell apoptosis and G0/G1 cell cycle arrest. This work functionally described the first ROP kinase from E. tenella and its non-canonical structure. Our study provides the first mechanistic insight into host cell apoptosis inhibition by E. tenella. EtROP1 appears as a new candidate for coccidiosis control.

International audience; Avian uterine fluid (UF) and uterovaginal sperm storage tubules (SST) are key components in accepting sperm in SST, maintaining sperm function for several weeks, releasing sperm from SST and their ascent through the uterus. To improve understanding of sperm storage processes requires investigating UF and SST. This study aimed to identify proteins modulated by sperm in the hen’s genital tract, and to highlight their role during sperm storage. Two genetic lines of hens exhibiting long (F+) or short (F-) sperm storage ability were used. GeLC MS/MS analysis was used to establish a quantitative inventory of proteins regulated after insemination in both lines. The proteomic data are available via ProteomeXchange with identifier PXD013514. Immunohistochemistry was used to identify high (ANXA4/ANXA5/OCX32) and low (HSPA8/PIGR) fertility markers in the uterovaginal junction. Our results demonstrated that sperm induced a significant and rapid change in the UF proteomic content, and also in the SST epithelium. In F+ hens, mobilization of the ANXA4 protein in the apical part of SST cells after insemination was associated with increased levels of some proteoglycans and binding proteins, and also antimicrobial eggshell matrix protein (OCX32) in the UF. We also observed increased levels of lipid transporters involved in egg formation (VTG1-2, APOA1-4-H). In F- hens, insemination induced increased levels of PIGR in both UF and SST, of ANXA5 in SST, of UF enzymes exhibiting metallopeptidase activity, and mucins. In conclusion, sperm induced significant changes in the UF proteomic content. This study also provides evidence that the SST immune system plays a major role in regulating sperm storage.

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].

International audience; In many amniotes, the amniotic fluid is depicted as a dynamic milieu that participates in the protection of the embryo (cushioning, hydration, and immunity). However, in birds, the protein profile of the amniotic fluid remains unexplored, even though its proteomic signature is predicted to differ compared with that of humans. In fact, unlike humans, chicken amniotic fluid does not collect excretory products and its protein composition strikingly changes at mid-development because of the massive inflow of egg white proteins, which are thereafter swallowed by the embryo to support its growth. Using GeLC-MS/MS and shotgun strategies, we identified 91 nonredundant proteins delineating the chicken amniotic fluid proteome at day 11 of development, before egg white transfer. These proteins were essentially associated with the metabolism of nutrients, immune response and developmental processes. Forty-eight proteins were common to both chicken and human amniotic fluids, including serum albumin, apolipoprotein A1 and alpha-fetoprotein. We further investigated the effective role of chicken amniotic fluid in innate defense and revealed that it exhibits significant antibacterial activity at day 11 of development. This antibacterial potential is drastically enhanced after egg white transfer, presumably due to lysozyme, avian beta-defensin 11, vitelline membrane outer layer protein 1, and beta-microseminoprotein-like as the most likely antibacterial candidates. Interestingly, several proteins recovered in the chicken amniotic fluid prior and after egg white transfer are uniquely found in birds (ovalbumin and related proteins X and Y, avian beta-defensin 11) or oviparous species (vitellogenins 1 and 2, riboflavin-binding protein). This study provides an integrative overview of the chicken amniotic fluid proteome and opens stimulating perspectives in deciphering the role of avian egg-specific proteins in embryonic development, including innate immunity. These proteins may constitute valuable biomarkers for poultry production to detect hazardous situations (stress, infection, etc.), that may negatively affect the development of the chicken embryo.

Overfed hens selected for their rapid growth become fatter and develop reproductive disorders. Herein, we aimed to demonstrate that food restriction leading to a weight reduction and/or a supplementation with fish oil may be effective in preventing reproductive disorders through the regulation of adipokine expression in broiler hens. This study included four groups of food restricted (Rt) orad libitumhens (Ad, feeding at a rate 1.7 times greater than Rt hens) supplemented or unsupplemented with fish oil (1%). The Rt diet significantly increased plasma chemerin (RARRES2) levels during the laying period, delayed sexual maturity by one week and improved egg quality and fertility. These effects were associated with higher progesterone production in response to IGF1 (or LH) in cultured granulosa cells andin vivoegg yolk, as compared with Ad hens. Fish oil supplementation had similar effects to the Rt diet on progesterone (P RARRES2levels were lower in theca cells of Rt hens andNAMPTlevels were increased by the fish oil supplementation. A significant positive correlation betweenRARRES2expression in granulosa cells and the weight of F1 preovulatory follicle was observed, as well as a negative correlation of plasma RARRES2 levels with hatchability. Thus, food restriction but not fish oil supplementation improved fertility, and this was associated with variations in RARRES2 plasma and ovarian expression in hens.

Background Genomic loci associated with histone marks are typically analyzed by immunoprecipitation of the chromatin followed by quantitative-PCR (ChIP-qPCR) or high throughput sequencing (ChIP-seq). Chromatin can be either cross-linked (X-ChIP) or used in the native state (N-ChIP). Cross-linking of DNA and proteins helps stabilizing their interactions before analysis. Despite X-ChIP is the most commonly used method, muscle tissue fixation is known to be relatively inefficient. Moreover, no protocol described a simple and reliable preparation of skeletal muscle chromatin of sufficient quality for subsequent high-throughput sequencing. Here we aimed to set-up and compare both chromatin preparation methods for a genome-wide analysis of H3K27me3, a broad-peak histone mark, using chicken P. major muscle tissue. Results Fixed and unfixed chromatin were prepared from chicken muscle tissues (Pectoralis major). Chromatin fixation, shearing by sonication or digestion and immunoprecipitation performed equivalently. High-quality Illumina reads were obtained (q30 > 93%). The bioinformatic analysis of the data was performed using epic, a tool based on SICER, and MACS2. Forty millions of reads were analyzed for both X-ChIP-seq and N-ChIP-seq experiments. Surprisingly, H3K27me3 X-ChIP-seq analysis led to the identification of only 2000 enriched regions compared to about 15,000 regions identified in the case of N-ChIP-seq. N-ChIP-seq peaks were more consistent between replicates compared to X-ChIP-seq. Higher N-ChIP-seq enrichments were confirmed by ChIP-qPCR at the PAX5 and SOX2 loci known to be enriched for H3K27me3 in myotubes and at the loci of common regions of enrichment identified in this study. Conclusions Our findings suggest that the preparation of muscle chromatin for ChIP-seq in cross-linked conditions can compromise the systematic analysis of broad histone marks. Therefore, native chromatin preparation should be preferred to cross-linking when a ChIP experiment has to be performed on skeletal muscle tissue, particularly when a broad source signal is considered. Electronic supplementary material The online version of this article (doi:10.1186/s12575-017-0059-0) contains supplementary material, which is available to authorized users.

International audience; To better appreciate the dynamics of yolk proteins during embryonic development, we analyzed the protein quantitative changes occurring in the yolk plasma at the day of lay and after 12 days of incubation, by comparing unfertilized and fertilized chicken eggs. Of the 127 identified proteins, 69 showed relative abundance differences among conditions. Alpha-fetoprotein and two uncharacterized proteins (F1NHB8 and F1NMM2) were identified for the first time in the egg. After 12 days of incubation, five proteins (vitronectin, alpha-fetoprotein, similar to thrombin, apolipoprotein B, and apovitellenin-I) showed a major increase in relative abundance, whereas 15 proteins showed a significant decrease in the yolks of fertilized eggs. In unfertilized/table eggs, we observed an accumulation of proteins likely to originate from other egg compartments during incubation. This study provides basic knowledge on the utilization of egg yolk proteins by the embryo and gives some insight into how storage can affect egg quality.

Background: Ovalbumin-related protein X (OVAX) is an uncharacterized ovalbumin-serpin. Results: This egg white-specific serpin lacks protease inhibitory activity, but unlike its ovalbumin homolog, OVAX exhibits antibacterial properties, partly through its heparin-binding site(s). Conclusion: OVAX, a non-inhibitory serpin is a heparin-binding molecule with antibacterial activity. Significance: OVAX participates in egg defense and constitutes a natural agent against Listeria and Salmonella., Ovalbumin family contains three proteins with high sequence similarity: ovalbumin, ovalbumin-related protein Y (OVAY), and ovalbumin-related protein X (OVAX). Ovalbumin is the major egg white protein with still undefined function, whereas the biological activity of OVAX and OVAY has not yet been explored. Similar to ovalbumin and OVAY, OVAX belongs to the ovalbumin serine protease inhibitor family (ov-serpin). We show that OVAX is specifically expressed by the magnum tissue, which is responsible for egg white formation. OVAX is also the main heparin-binding protein of egg white. This glycoprotein with a predicted reactive site at Lys367-His368 is not able to inhibit trypsin, plasmin, or cathepsin G with or without heparin as a cofactor. Secondary structure of OVAX is similar to that of ovalbumin, but the three-dimensional model of OVAX reveals the presence of a cluster of exposed positive charges, which potentially explains the affinity of this ov-serpin for heparin, as opposed to ovalbumin. Interestingly, OVAX, unlike ovalbumin, displays antibacterial activities against both Listeria monocytogenes and Salmonella enterica sv. Enteritidis. These properties partly involve heparin-binding site(s) of the molecule as the presence of heparin reverses its anti-Salmonella but not its anti-Listeria potential. Altogether, these results suggest that OVAX and ovalbumin, although highly similar in sequence, have peculiar sequential and/or structural features that are likely to impact their respective biological functions.

Background Meat type chickens have limited capacities to cope with high environmental temperatures, this sometimes leading to mortality on farms and subsequent economic losses. A strategy to alleviate this problem is to enhance adaptive capacities to face heat exposure using thermal manipulation (TM) during embryogenesis. This strategy was shown to improve thermotolerance during their life span. The aim of this study was to determine the effects of TM (39.5 °C, 12 h/24 vs 37.8 °C from d7 to d16 of embryogenesis) and of a subsequent heat challenge (32 °C for 5 h) applied on d34 on gene expression in the Pectoralis major muscle (PM). A chicken gene expression microarray (8 × 60 K) was used to compare muscle gene expression profiles of Control (C characterized by relatively high body temperatures, Tb) and TM chickens (characterized by a relatively low Tb) reared at 21 °C and at 32 °C (CHC and TMHC, respectively) in a dye-swap design with four comparisons and 8 broilers per treatment. Real-time quantitative PCR (RT-qPCR) was subsequently performed to validate differential expression in each comparison. Gene ontology, clustering and network building strategies were then used to identify pathways affected by TM and heat challenge. Results Among the genes differentially expressed (DE) in the PM (1.5 % of total probes), 28 were found to be differentially expressed between C and TM, 128 between CHC and C, and 759 between TMHC and TM. No DE gene was found between TMHC and CHC broilers. The majority of DE genes analyzed by RT-qPCR were validated. In the TM/C comparison, DE genes were involved in energy metabolism and mitochondrial function, cell proliferation, vascularization and muscle growth; when comparing heat-exposed chickens to their own controls, TM broilers developed more specific pathways than C, especially involving genes related to metabolism, stress response, vascularization, anti-apoptotic and epigenetic processes. Conclusions This study improved the understanding of the long-term effects of TM on PM muscle. TM broilers displaying low Tb may have lower metabolic intensity in the muscle, resulting in decreased metabolic heat production, whereas modifications in vascularization may enhance heat loss. These specific changes could in part explain the better adaptation of TM broilers to heat. Electronic supplementary material The online version of this article (doi:10.1186/s12864-016-2661-y) contains supplementary material, which is available to authorized users.

Remerciements : GenoToul, Plateforme Bioinformatique de Toulouse Midi-Pyrénées; International audience; Eggshell is a bioceramic composed of 95% calcium carbonate mineral and 3.5% organic matrix. Its structural organisation is controlled by its organic matrix. We have used quantitative proteomics to study four key stages of shell mineralisation: 1) widespread deposition of amorphous calcium carbonate (ACC), 2) ACC transformation into crystalline calcite aggregates, 3) formation of larger calcite crystal units 4) and development of a columnar structure with preferential calcite crystal orientation. This approach explored the distribution of 216 shell matrix proteins found at the four stages. Variations in abundance according to these calcification events were observed for 175 proteins. A putative function related to the mineralisation process was predicted by bioinformatics for 77 of them and was further characterised. We confirmed the important role of lysozyme, ovotransferrin, ovocleidin-17 and ovocleidin-116 for shell calcification process, characterised major calcium binding proteins (EDIL3, ALB, MFGE8, NUCB2), and described novel proteoglycans core proteins (GPC4, HAPLN3). We suggest that OVAL and OC-17 play a role in the stabilisation of ACC. Finally, we report proteins involved in the regulation of proteins driving the mineralisation. They correspond to numerous molecular chaperones including CLU, PPIB and OCX21, protease and protease inhibitors including OVM and CST3, and regulators of phosphorylation. Significance The avian eggshell is a natural protective barrier of the egg as long as it remains intact. It prevents microbial penetration to ensure a harmonious development of the embryo and table eggs free of pathogens for consumers. The eggshell is a calcitic biomineral structure initiated on organic nucleation sites which are evenly disposed on eggshell membranes. Its structural organisation results from an interaction between matrix proteins and calcium carbonate, in particular calcite crystal during the growth phase. The initial steps of the calcification process during which calcitic mineral phase is selected and the process of crystal orientation selection by competition for space are crucial in the determination of the whole eggshell columnar ultrastructure and its resulting exceptional mechanical properties. Recent studies have also described the physical characteristics of calcium carbonate during the initial sequence of calcium carbonate biomineralisation in many species and have highlighted the key role of amorphous calcium carbonate. In order to determine precisely the importance of particular proteins relative to these key events at the onset of mineralisation, this project used quantitative proteomics of matrix proteins in forming eggshell, assuming that a protein more abundant at a particular stage of the process has an higher probability to be involved in its control. This information was coupled with bioinformatic analysis predicting the function of proteins in the stabilisation of disordered forms of calcium carbonate and their influence on crystal polymorph and morphology. Eggshell calcification is a relevant model for studying calcium carbonate biomineralisation on a membrane support. Better understanding of this process will provide insight into the fabrication of ceramics at ambient pressures and temperatures and might provide means of reinforcing its solidity.

Background In Gallus gallus, eggshell formation takes place daily in the hen uterus and requires large amounts of the ionic precursors for calcium carbonate (CaCO3). Both elements (Ca2+, HCO3 -) are supplied by the blood via trans-epithelial transport. Our aims were to identify genes coding for ion transporters that are upregulated in the uterine portion of the oviduct during eggshell calcification, compared to other tissues and other physiological states, and incorporate these proteins into a general model for mineral transfer across the tubular gland cells during eggshell formation. Results A total of 37 candidate ion transport genes were selected from our database of overexpressed uterine genes associated with eggshell calcification, and by analogy with mammalian transporters. Their uterine expression was compared by qRTPCR in the presence and absence of eggshell formation, and with relative expression levels in magnum (low Ca2+/HCO3 - movement) and duodenum (high rates of Ca2+/HCO3 - trans-epithelial transfer). We identified overexpression of eleven genes related to calcium movement: the TRPV6 Ca2+ channel (basolateral uptake of Ca2+), 28 kDa calbindin (intracellular Ca2+ buffering), the endoplasmic reticulum type 2 and 3 Ca2+ pumps (ER uptake), and the inositol trisphosphate receptors type 1, 2 and 3 (ER release). Ca2+ movement across the apical membrane likely involves membrane Ca2+ pumps and Ca2+/Na+ exchangers. Our data suggests that Na+ transport involved the SCNN1 channel and the Na+/Ca2+ exchangers SLC8A1, 3 for cell uptake, the Na+/K+ ATPase for cell output. K+ uptake resulted from the Na+/K+ ATPase, and its output from the K+ channels (KCNJ2, 15, 16 and KCNMA1). We propose that the HCO3 - is mainly produced from CO2 by the carbonic anhydrase 2 (CA2) and that HCO3 - is secreted through the HCO3 -/Cl- exchanger SLC26A9. HCO3 - synthesis and precipitation with Ca2+ produce two H+. Protons are absorbed via the membrane’s Ca2+ pumps ATP2B1, 2 in the apical membrane and the vacuolar (H+)-atpases at the basolateral level. Our model incorporate Cl- ions which are absorbed by the HCO3 -/Cl- exchanger SLC26A9 and by Cl- channels (CLCN2, CFTR) and might be extruded by Cl-/H+ exchanger (CLCN5), but also by Na+ K+ 2 Cl- and K+ Cl- cotransporters. Conclusions Our Gallus gallus uterine model proposes a large list of ion transfer proteins supplying Ca2+ and HCO3 - and maintaining cellular ionic homeostasis. This avian model should contribute towards understanding the mechanisms and regulation for ionic precursors of CaCO3, and provide insight in other species where epithelia transport large amount of calcium or bicarbonate.

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Because of prolonged sperm storage in their oviduct, domestic hens can produce fertile eggs for up to 3 weeks following a single AI. The oviduct secretions may have an effect on sperm survival, but its composition during fertilization is unknown. In the present study, we compared the proteomic content of uterine fluid collected from two lines of hens divergent by their duration of fertility period (DFP), which defined sperm-storage duration. The first line displays a shorter period of sperm storage (10 days, line DFP–), whereas the second displays a longer period of sperm storage (21 days, DFP+). The aim was to identify proteins or peptides that may be involved in spermatozoa survival. Uterine fluid was collected 10 h after oviposition either before (n = 5/line) or 24 h after (n = 5/line) AI. Samples were pooled by condition: DFP+ before AI, DFP+ after AI, DFP– before AI, and DFP– after AI. Bottom-up approach using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and nano LC-MS/MS was performed (3 replicates). Data were matched against the NCBInr database (2014) using Mascot, and identifications were validated by the peptide and protein Prophet algorithm using Scaffold software. To determine the differences in protein expression, spectral counting and XIC quantitative methods were employed using Scaffold Q+ (P 2). Two proteins were up-regulated and one was down-regulated in oviductal secretion of both lines in response to AI. However, AI induced a significantly different abundance between protein content of DFP– and DFP+ fluids. A panel of 8 proteins, included one DFP+-specific protein, was more abundant in DFP+ line than in DFP–. Only one protein was less abundant in DFP+ line than in DFP–. In conclusion, the presence of sperm in the genital tract induced quantitative differences of the protein content of the uterine fluid in DFP– and DFP+ hen lines. These differences imply proteins that are known as male proteins (sperm, seminal plasma, testis). Analysis of sperm protein modifications after storage will help us to understand the functional implication of these candidates.

Chicken eggshell is a biomineral composed of 95% calcite calcium carbonate mineral and of 3.5% organic matrix proteins. The assembly of mineral and its structural organization is controlled by its organic matrix. In a recent study [1], we have used quantitative proteomic, bioinformatic and functional analyses to explore the distribution of 216 eggshell matrix proteins at four key stages of shell mineralization defined as: (1) widespread deposition of amorphous calcium carbonate (ACC), (2) ACC transformation into crystalline calcite aggregates, (3) formation of larger calcite crystal units and (4) rapid growth of calcite as columnar structure with preferential crystal orientation. The current article detailed the quantitative analysis performed at the four stages of shell mineralization to determine the proteins which are the most abundant. Additionally, we reported the enriched GO terms and described the presence of 35 antimicrobial proteins equally distributed at all stages to keep the egg free of bacteria and of 81 proteins, the function of which could not be ascribed.

Background 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. Results 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 (α-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 α-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. Conclusions 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. Electronic supplementary material The online version of this article (doi:10.1186/s12864-015-2013-3) contains supplementary material, which is available to authorized users.

International audience; Context: Coccidia are obligate intracellular protozoan parasites responsible for human and veterinary diseases. Eimeria tenella, the etiologic agent of cecal coccidiosis, is a major pathogen of chickens. In Toxoplasma gondii, some kinases from the rhoptry compartment (ROP) are key virulence factors. ROP kinases hijack and modulate many cellular functions and pathways, allowing T. gondii survival and development. E. tenella's kinome comprises 28 putative members of the ROP kinase family; most of them are predicted, as pseudokinases and their functions have never been characterized. One of the predicted kinase, EtROP1, was identified in the rhoptry proteome of E. tenella sporozoites. Here, we demonstrated that EtROP1 is active, and the N‐terminal extension is necessary for its catalytic kinase activity. Methods and results: Ectopic expression of EtROP1 followed by co‐immunoprecipitation identified cellular p53 as EtROP1 partner. Further characterization confirmed the interaction and the phosphorylation of p53 by EtROP1. E. tenella infection or overexpression of EtROP1 resulted both in inhibition of host cell apoptosis and G0/G1 cell cycle arrest.Conclusions: This work functionally described the first ROP kinase from E. tenella and its non-canonical structure. Our study provides the first mechanistic insight into the inhibition of host cell apoptosis by E. tenella. EtROP1 appears as a new candidate for coccidiosis control.

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