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The Unique Features of Proteins Depicting the Chicken Amniotic Fluid*

Open AccessPublished:February 14, 2018DOI:https://doi.org/10.1074/mcp.RA117.000459
      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.
      In oviparous species, embryonic development depends on the various components, nutrients and structures composing the eggshell, the egg yolk, the egg white and the vitelline membrane (
      • Moran Jr, E.T.
      Nutrition of the developing embryo and hatchling.
      ). It also relies on the proper development of the extra-embryonic structures, namely the yolk sac, the amniotic sac and the allantoic/chorioallantoic sac (
      • Moran Jr, E.T.
      Nutrition of the developing embryo and hatchling.
      ) (Fig. 1A,). These structures develop at the very early stages of development and originate from embryonic tissues but are not considered to be part of the embryonic body (
      • Bellairs R.
      • Osmond M.
      ). They are discarded or resorbed at hatching. These living structures are partly preserved among amniote species, but exhibit evolutionary particularities depending on the embryonic development mode (
      • Sheng G.
      • Foley A.C.
      Diversification and conservation of the extraembryonic tissues in mediating nutrient uptake during amniote development.
      ). The yolk sac, which appears in the first stages of development, degenerates rapidly in mammals (Fig. 1B,), whereas in some birds, it participates in digestive processes until the last stages of incubation prior to complete abdominal resorption at hatch. The yolk sac may have many other functions, which are temporally regulated during incubation: it resembles the liver in the synthesis of plasma proteins, the bone marrow in erythropoiesis, and the intestine, in digestion of nutrients and their transport to the embryo (
      • Yadgary L.
      • Wong E.A.
      • Uni Z.
      Temporal transcriptome analysis of the chicken embryo yolk sac.
      ). Thus, the yolk sac plays different roles to support or replace the functions of several organs that have not yet reached their full functional capacity. The chorioallantoic sac is composed of the chorioallantoic membrane, which results from the fusion of the chorion and the allantois at day 5/6 of incubation (ED5/ED6), and it includes the allantoic fluid. It is a highly vascularized structure that performs many functions during chicken embryonic development: it collects nitrogenous and excretory products from the embryonic metabolism, it participates in respiratory exchange, in calcium transport from the eggshell toward the embryo, in ion and water reabsorption from the allantoic fluid and, thus, in acid-base homeostasis (
      • Bellairs R.
      • Osmond M.
      ,
      • Gabrielli M.G.
      • Accili D.
      The chick chorioallantoic membrane: a model of molecular, structural, and functional adaptation to transepithelial ion transport and barrier function during embryonic development.
      ). In humans, the allantoic sac forms only a part of the umbilical cord. Concerning the amniotic sac, it is described in all amniotes as a structure, with amniotic fluid (AF)
      The abbreviations used are: AF, Amniotic fluid; ATCC, American Type Culture Collection; BMSP, Beta-microseminoprotein-like; ED, Day of incubation; emPAI, Exponentially modified Protein Abundance Index; EW, Egg white; HBP, Heparin-binding protein; L.m., Listeria monocytogenes,; MYA, Million Years Ago; NCBI, National Center for Biotechnology Information; S,. E., Salmonella enterica, Enteritidis; TSB, Trypticase soy broth.
      1The abbreviations used are: AF, Amniotic fluid; ATCC, American Type Culture Collection; BMSP, Beta-microseminoprotein-like; ED, Day of incubation; emPAI, Exponentially modified Protein Abundance Index; EW, Egg white; HBP, Heparin-binding protein; L.m., Listeria monocytogenes,; MYA, Million Years Ago; NCBI, National Center for Biotechnology Information; S,. E., Salmonella enterica, Enteritidis; TSB, Trypticase soy broth.
      , which protects the embryo against mechanical shocks, dehydration or adhesion to the other extra-embryonic membranes. It also serves as a source of nutrients (
      • Underwood M.A.
      • Gilbert W.M.
      • Sherman M.P.
      Amniotic fluid: not just fetal urine anymore.
      ). It provides a favorable environment for the development of the embryo: pH of about 7.1 to 7.3, stable temperature, and sensorial stimulation (taste, sense of smell and hearing) (
      • Bakalar N.
      Sensory science: partners in flavour.
      ).
      Figure thumbnail gr1
      Fig. 1.Schematic representation of the extraembryonic structures during the chicken (A,) and human embryonic development (B,); chicken embryo at mid-incubation (11 days) and human embryo at mid-gestation (21 weeks), respectively.
      Human AF is a fluctuating milieu mainly composed of water (about 96.4%), minerals, trace elements, carbohydrates, hormones, glucose, lipids, urea, cells, free amino-acids, proteins and peptides (
      • Underwood M.A.
      • Gilbert W.M.
      • Sherman M.P.
      Amniotic fluid: not just fetal urine anymore.
      ,
      • Orczyk-Pawilowicz M.
      • Jawien E.
      • Deja S.
      • Hirnle L.
      • Zabek A.
      • Mlynarz P.
      Metabolomics of human amniotic fluid and maternal plasma during normal pregnancy.
      ). Its biochemical composition changes with gestational age/developmental stage as a result of various physiological mechanisms including feto-maternal exchanges: AF swallowing and lung fluid production by the embryo, excretion of fetal urine and transfer of solutes and fluids across amniotic and uterine membranes (Fig. 1B,) and across skin especially before keratinization (
      • Underwood M.A.
      • Gilbert W.M.
      • Sherman M.P.
      Amniotic fluid: not just fetal urine anymore.
      ,
      • Michaels J.E.A.
      • Dasari S.
      • Pereira L.
      • Reddy A.P.
      • Lapidus J.A.
      • Lu X.F.
      • Jacob T.
      • Thomas A.
      • Rodland M.
      • Roberts C.T.
      • Gravett M.G.
      • Nagalla S.R.
      Comprehensive proteomic analysis of the human amniotic fluid proteome: Gestational age-dependent changes.
      ). In humans, before skin keratinization (25 weeks of age), the composition of the AF is very similar to fetal blood plasma. As a dynamic milieu reflecting the physiological or pathological status of the embryo, the biochemical composition of the AF has been extensively characterized in humans since it contains molecular markers for the detection of embryonic abnormal development, inflammation, infection or pregnancy-related complications (
      • Romero R.
      • Kusanovic J.P.
      • Gotsch F.
      • Erez O.
      • Vaisbuch E.
      • Mazaki-Tovi S.
      • Moser A.
      • Tam S.
      • Leszyk J.
      • Master S.R.
      • Juhasz P.
      • Pacora P.
      • Ogge G.
      • Gomez R.
      • Yoon B.H.
      • Yeo L.
      • Hassan S.S.
      • Rogers W.T.
      Isobaric labeling and tandem mass spectrometry: A novel approach for profiling and quantifying proteins differentially expressed in amniotic fluid in preterm labor with and without intra-amniotic infection/inflammation.
      ). Its role as a mechanical cushioning of the embryo, together with the fact that 25% of its total protein content is associated with the immune response or is related to defense (
      • Michaels J.E.A.
      • Dasari S.
      • Pereira L.
      • Reddy A.P.
      • Lapidus J.A.
      • Lu X.F.
      • Jacob T.
      • Thomas A.
      • Rodland M.
      • Roberts C.T.
      • Gravett M.G.
      • Nagalla S.R.
      Comprehensive proteomic analysis of the human amniotic fluid proteome: Gestational age-dependent changes.
      ), corroborate its primary role in the protection of the embryo. However, it seems that proteins of the human AF are not solely involved in the nutrition and protection of the embryo, but they display many other functions related to metabolism and development (
      • Cho C.K.J.
      • Shan S.J.
      • Winsor E.J.
      • Diamandis E.P.
      Proteomics analysis of human amniotic fluid.
      ). In oviparous species, however, the functions of the AF are still poorly understood and there are several structural and biochemical particularities that suggest similar but also diverging functions. First, there are two major extraembryonic fluids in birds, the amniotic and allantoic fluids, which are physically separated, to ensure different functions. Like humans, the bird AF is contained in the amniotic sac and bathes the embryo, whereas the allantoic fluid is secreted in the chorioallantoic sac (Fig. 1A,), which is an intestinal intussusception of the embryo that receives disposable wastes directly from the embryonic kidneys. These anatomical specificities deeply influence the biochemical composition of the AF, which thus in birds does not collect fetal urine. The second main difference, as compared with mammals, is that after ED12, egg white proteins are massively transferred into the amniotic sac (
      • Yoshizaki N.
      • Ito Y.
      • Hori H.
      • Saito H.
      • Iwasawa A.
      Absorption, transportation and digestion of egg white in quail embryos.
      ), where they are absorbed orally by the embryo as a source of amino acids to support its rapid growth (
      • Geelhoed S.E.
      • Conklin J.L.
      An electrophoretic study of proteins in chick embryonic fluids.
      ,
      • Baintner K.
      • Fehér G.
      Fate of egg white trypsin inhibitor and start of proteolysis in developing chick embryo and newly hatched chick.
      ,
      • Cirkvenčič N.
      • Narat M.
      • Dovč P.
      • Benčina D.
      Distribution of chicken cathepsins B and L, cystatin and ovalbumin in extra-embryonic fluids during embryogenesis.
      ,
      • Sugimoto Y.
      • Sanuki S.
      • Ohsako S.
      • Higashimoto Y.
      • Kondo M.
      • Kurawaki J.
      • Ibrahim H.R.
      • Aoki T.
      • Kusakabe T.
      • Koga K.
      Ovalbumin in developing chicken eggs migrates from egg white to embryonic organs while changing its conformation and thermal stability.
      ,
      • Muramatsu T.
      • Hiramoto K.
      • Koshi N.
      • Okumura J.
      • Miyoshi S.
      • Mitsumoto T.
      Importance of albumen content in whole-body protein synthesis of the chicken embryo during incubation.
      ) until hatching (ED21). Consequently, this process drastically impacts the protein concentration of the AF, which is barely measurable before the 11th day of incubation (ED11) (about 0.01 mg/ml) and reaches 200 mg/ml following egg white transfer (
      • Da Silva M.
      • Labas V.
      • Nys Y.
      • Rehault-Godbert S.
      Investigating proteins and proteases composing amniotic and allantoic fluids during chicken embryonic development.
      ). In fact, before ED11, the chicken AF is mainly composed of water and mineral elements, such as chloride, sodium, potassium, phosphorus, magnesium, calcium, iron, and sulfur, like human AF (
      • Romanoff A.L.
      ,
      • Romanoff A.L.
      • Romanoff A.J.
      ). More recently, a study analyzing the major proteins of the chicken AF, revealed that it contains egg white proteins even before the massive egg white transfer at ED12 (
      • Da Silva M.
      • Labas V.
      • Nys Y.
      • Rehault-Godbert S.
      Investigating proteins and proteases composing amniotic and allantoic fluids during chicken embryonic development.
      ). Some of these proteins are egg yolk proteins while others may originate from the embryo (skin, feather) or its extra-embryonic membranes (amniotic, yolk, and chorioallantoic sacs). All these proteins have been associated with functions comparable to those described for human AF, including metabolism, immune system or tissue remodeling, but they also serve as a major source of amino acids and energy for the embryo, especially during the second half of incubation. The avian specificity of some egg white and yolk proteins (
      • Tian X.
      • Gautron J.
      • Monget P.
      • Pascal G.
      What Makes an Egg Unique? Clues from Evolutionary Scenarios of Egg-Specific Genes.
      ) that are recovered in the AF, together with the physical separation of amniotic sac from the embryonic urinary system (chorioallantoic sac) and the transfer of egg white proteins at midincubation, suggest that this fluid may have very specific biological functions related to birds or even oviparity.
      In such a stimulating context, we explored the avian AF proteins and specificities using the chicken as a model of birds. Similar to human AF, with albumin, immunoglobulins, transferrin and haptoglobin (
      • Michaels J.E.A.
      • Dasari S.
      • Pereira L.
      • Reddy A.P.
      • Lapidus J.A.
      • Lu X.F.
      • Jacob T.
      • Thomas A.
      • Rodland M.
      • Roberts C.T.
      • Gravett M.G.
      • Nagalla S.R.
      Comprehensive proteomic analysis of the human amniotic fluid proteome: Gestational age-dependent changes.
      ,
      • Cho C.K.J.
      • Shan S.J.
      • Winsor E.J.
      • Diamandis E.P.
      Proteomics analysis of human amniotic fluid.
      ), chicken AF contains a few major proteins (ovalbumin, ovotransferrin, etc.) that complicate proteome profiling and mask the presence of proteins of lower abundance. Therefore, we designed a bottom-up proteomic approach combining two complementary strategies as GeLC-MS/MS (protein samples fractionated by SDS-PAGE are analyzed by nanoLC-MS/MS after in-gel digestion) and shotgun (protein samples are directly analyzed by nanoLC-MS/MS after in-solution digestion) to give an exhaustive view of the AF proteome at ED11, before the substantial transfer of egg white (which completely changes the global protein profile). We performed a functional annotation of AF proteome using Gene Ontology approaches: 1) on the 10 most abundant proteins representing 66% to 81% of the total protein contents for GeLC-MS/MS and shotgun analyses, respectively, and 2) on the complete list of proteins resulting from both analyses. This approach was compulsory because many of the major proteins composing egg structures do not have assigned functions in databases yet and consequently, the functional annotation of the entire list would enrich known functions, whose biological significance may be overestimated. Considering the importance of human amniotic fluid in innate immunity and to better appreciate the contribution of chicken amniotic fluid to embryo defense against microorganisms, we used an in-gel antibacterial assay combined to mass spectrometry, to identify antibacterial proteins and peptides contained in AF or in enriched fractions of AF, before and after egg white transfer. As the protein composition of the chicken AF is temporally regulated during incubation, AF functions are thoroughly modified following egg white transfer. Finally, a comparison with the published proteomes of the human AF (
      • Cho C.K.J.
      • Shan S.J.
      • Winsor E.J.
      • Diamandis E.P.
      Proteomics analysis of human amniotic fluid.
      ) and a phylogenetic study on all proteins identified in this study, allowed us to highlight chicken AF specificities, revealing a set of proteins, which are only present in birds and whose physiological roles remain fully opened.

      EXPERIMENTAL PROCEDURES

      A diagram describing the experimental design is presented in Fig. 2.
      Figure thumbnail gr2
      Fig. 2.Diagram describing the experimental design. A, preparation of biological samples, B, proteomic analyses using Shotgun and GeLC-MS/MS analyses, C, antibacterial potential of egg samples and identification of antibacterial candidates. AF, amniotic fluid; Cb, Coomassie Brilliant Blue staining; ED, Day of incubation; EW, egg white; HBP, heparin-binding proteins.

      Samplings

      Fluids were sampled as previously described by Da Silva et al., (
      • Da Silva M.
      • Labas V.
      • Nys Y.
      • Rehault-Godbert S.
      Investigating proteins and proteases composing amniotic and allantoic fluids during chicken embryonic development.
      ). Briefly, fertile eggs were incubated under standard conditions (45% relative humidity, 37.8 °C, automatic turning every hour), after a three-day storage at 16 °C and 85% relative humidity to ensure synchronization of developmental stages (UE1295, INRA, F-37380 Nouzilly, France). At ED11 and ED16, 40 eggs of comparable weight (62.4 ± 4.3 g) containing viable embryos (checked by candling) were tested with the Acoustic Egg Tester (KU Leuven, Belgium), and cracked eggs were discarded. By ED11, egg white was sampled with a syringe after drilling a hole in the eggshell. After removing the eggshell, the egg content was poured into a Petri dish and the AF was recovered with a syringe through the amniotic membrane. By ED16, the egg white was collected in the Petri dish with pliers due to its high viscosity. All samplings were performed under sterile conditions. Sex of individual embryos was determined using PCR (
      • Clinton M.
      • Haines L.
      • Belloir B.
      • McBride D.
      Sexing chick embryos: a rapid and simple protocol.
      ).

      Fluid Characterization

      After samplings, AF were centrifuged at 3,000 g (10 min, 4 °C) to remove insoluble components. Volumes, pH (Microelectrode pH InLab 423, Fisher Scientific, Illkirch, France), osmolality (Fiske Mark 3 Osmometer, Advanced Instruments, Niederbronn-Les-Bains, France), and absorbance spectrum (Nanodrop, ND-100 Spectro, Wilmington, USA) were analyzed for each sample. The total protein concentration for each sample was assessed using BioRad DC Protein Assay Kit II (BioRad, Marnes-la-Coquette, France). All samples (25 μl) containing loading buffer (5X loading buffer: 0.25 m Tris-HCl, 0.05% bromphenol blue, 50% glycerol, 5% SDS, pH 6.8) were independently loaded on a 12.5% SDS-PAGE (1 mm) using a Mini-Protean II electrophoresis cell (BioRad), and further stained with Coomassie Brilliant Blue G250 or silver nitrate. This overall characterization (pH, osmolality, protein concentration, absorbance and electrophoretic patterns, and embryo's sex) helped us to select homogenous samples that were stored at − 20 °C for further analyses by mass spectrometry (supplemental Data S1).

      In-Gel and In-Solution Tryptic Digestion

      Twelve homogenous AF samples including six males and six females were pooled for protein identification. Proteins were either separated by a 4–20% SDS-PAGE followed by Coomassie Brilliant Blue G250 staining (GeLC-MS/MS analysis), or directly identified in solution (shotgun analysis) as described previously (
      • Da Silva M.
      • Labas V.
      • Nys Y.
      • Rehault-Godbert S.
      Investigating proteins and proteases composing amniotic and allantoic fluids during chicken embryonic development.
      ). The SDS-PAGE gel was cut in 20 sections (Fig. 3B,) and each slice was rinsed separately in water and then acetonitrile. Proteins were then reduced with dithiothreitol, alkylated with iodoacetamide, and incubated overnight at 37 °C in 25 mm NH4HCO3 with 12.5 ng/μl trypsin (Sequencing grade, Roche, Paris, France) as described by Shevchenko (
      • Shevchenko A.
      • Wilm M.
      • Vorm O.
      • Mann M.
      Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels.
      ). Peptides were pooled and dried using a SPD1010 speedvac system. Peptide mixtures associated to each band and in-solution samples were analyzed using nanoLC-MS/MS.
      Figure thumbnail gr3
      Fig. 3.SDS-PAGE analysis of the chicken amniotic fluid (AF) during incubation. A, 12.5% SDS-PAGE analysis of the AF from days 8–11 (ED8–11) to days 12–16 (ED12–16) of the incubation (2 μg and 10 μg, respectively) followed by Coomassie Brilliant Blue staining. B, 4–20% SDS-PAGE profile of AF at ED11 for GeLC-MS/MS analysis. Horizontal lanes and numbers indicate the position of gel slices (
      • Moran Jr, E.T.
      Nutrition of the developing embryo and hatchling.
      ,
      • Bellairs R.
      • Osmond M.
      ,
      • Sheng G.
      • Foley A.C.
      Diversification and conservation of the extraembryonic tissues in mediating nutrient uptake during amniote development.
      ,
      • Yadgary L.
      • Wong E.A.
      • Uni Z.
      Temporal transcriptome analysis of the chicken embryo yolk sac.
      ,
      • Gabrielli M.G.
      • Accili D.
      The chick chorioallantoic membrane: a model of molecular, structural, and functional adaptation to transepithelial ion transport and barrier function during embryonic development.
      ,
      • Underwood M.A.
      • Gilbert W.M.
      • Sherman M.P.
      Amniotic fluid: not just fetal urine anymore.
      ,
      • Bakalar N.
      Sensory science: partners in flavour.
      ,
      • Orczyk-Pawilowicz M.
      • Jawien E.
      • Deja S.
      • Hirnle L.
      • Zabek A.
      • Mlynarz P.
      Metabolomics of human amniotic fluid and maternal plasma during normal pregnancy.
      ,
      • Michaels J.E.A.
      • Dasari S.
      • Pereira L.
      • Reddy A.P.
      • Lapidus J.A.
      • Lu X.F.
      • Jacob T.
      • Thomas A.
      • Rodland M.
      • Roberts C.T.
      • Gravett M.G.
      • Nagalla S.R.
      Comprehensive proteomic analysis of the human amniotic fluid proteome: Gestational age-dependent changes.
      ,
      • Romero R.
      • Kusanovic J.P.
      • Gotsch F.
      • Erez O.
      • Vaisbuch E.
      • Mazaki-Tovi S.
      • Moser A.
      • Tam S.
      • Leszyk J.
      • Master S.R.
      • Juhasz P.
      • Pacora P.
      • Ogge G.
      • Gomez R.
      • Yoon B.H.
      • Yeo L.
      • Hassan S.S.
      • Rogers W.T.
      Isobaric labeling and tandem mass spectrometry: A novel approach for profiling and quantifying proteins differentially expressed in amniotic fluid in preterm labor with and without intra-amniotic infection/inflammation.
      ,
      • Cho C.K.J.
      • Shan S.J.
      • Winsor E.J.
      • Diamandis E.P.
      Proteomics analysis of human amniotic fluid.
      ,
      • Yoshizaki N.
      • Ito Y.
      • Hori H.
      • Saito H.
      • Iwasawa A.
      Absorption, transportation and digestion of egg white in quail embryos.
      ,
      • Geelhoed S.E.
      • Conklin J.L.
      An electrophoretic study of proteins in chick embryonic fluids.
      ,
      • Baintner K.
      • Fehér G.
      Fate of egg white trypsin inhibitor and start of proteolysis in developing chick embryo and newly hatched chick.
      ,
      • Cirkvenčič N.
      • Narat M.
      • Dovč P.
      • Benčina D.
      Distribution of chicken cathepsins B and L, cystatin and ovalbumin in extra-embryonic fluids during embryogenesis.
      ,
      • Sugimoto Y.
      • Sanuki S.
      • Ohsako S.
      • Higashimoto Y.
      • Kondo M.
      • Kurawaki J.
      • Ibrahim H.R.
      • Aoki T.
      • Kusakabe T.
      • Koga K.
      Ovalbumin in developing chicken eggs migrates from egg white to embryonic organs while changing its conformation and thermal stability.
      ,
      • Muramatsu T.
      • Hiramoto K.
      • Koshi N.
      • Okumura J.
      • Miyoshi S.
      • Mitsumoto T.
      Importance of albumen content in whole-body protein synthesis of the chicken embryo during incubation.
      ,
      • Da Silva M.
      • Labas V.
      • Nys Y.
      • Rehault-Godbert S.
      Investigating proteins and proteases composing amniotic and allantoic fluids during chicken embryonic development.
      ,
      • Romanoff A.L.
      ,
      • Romanoff A.L.
      • Romanoff A.J.
      ) prepared for in-gel digestion by trypsin.

      NanoLC-MS/MS

      The resulting peptide mixtures were analyzed using a LTQ Orbitrap Velos Mass Spectrometer (Thermo Fisher Scientific, Germany) coupled to an Ultimate® 3000 RSLC chromatographer controlled by Chromeleon 6.8 software (Dionex, Amsterdam, The Netherlands). Five microliters of sample were desalted and preconcentrated on a trap column (Acclaim PepMap 100 C18, 100 μm inner diameter × 2 cm long, 3 μm particles, 100 Å pores) for 10 min at 5 μl/min with 4% solvent B (0.1% formic acid, 15.9% water, 84% acetonitrile) in solvent A (0.1% formic acid, 97.9% water, 2% acetonitrile). Separation was conducted using a nanocolumn (Acclaim PepMap C18, 75 μm inner diameter × 50 cm long, 3 μm particles, 100 Å pores) at 300 nL/min by applying a gradient of 4 to 55% of solvent B during 90 min for GeLC-MS/MS analyses and during 120 min for shotgun analyses.
      Data were acquired using Xcalibur 2.1 software (Thermo Fisher Scientific). The instrument was operated in positive mode in data-dependent mode. Survey full scan MS spectra (from 400 to 1800 m,/z,) were acquired with a resolution set at 60,000. The 20 most intense ions with charge states ≥ 2 were sequentially isolated (isolation width: 2 m,/z,; 1 microscan) and fragmented using CID mode (energy of 35% and wideband-activation enabled). Dynamic exclusion was active during 30 s with a repeat count of one. Polydimethylcyclosiloxane (m,/z, 445.1200025) ions were used for internal calibration.
      The mass spectrometry proteomics data have been submitted to the ProteomeXchange Consortium and are available via, the PRIDE partner repository (
      • Vizcaino J.A.
      • Deutsch E.W.
      • Wang R.
      • Csordas A.
      • Reisinger F.
      • Rios D.
      • Dianes J.A.
      • Sun Z.
      • Farrah T.
      • Bandeira N.
      • Binz P.A.
      • Xenarios I.
      • Eisenacher M.
      • Mayer G.
      • Gatto L.
      • Campos A.
      • Chalkley R.J.
      • Kraus H.J.
      • Albar J.P.
      • Martinez-Bartolome S.
      • Apweiler R.
      • Omenn G.S.
      • Martens L.
      • Jones A.R.
      • Hermjakob H.
      ProteomeXchange provides globally coordinated proteomics data submission and dissemination.
      ) with the data set identifiers PXD008046 and 10.6019/PXD008046.

      Protein Identification and Data Validation

      The reliability between replicates was investigated by comparing chromatograms and spectra with Xcalibur 2.1 software. MS/MS ion searches were performed using Mascot search engine v 2.3.2 (Matrix Science, London, UK) via, Proteome Discoverer 2.1 software (ThermoFisher Scientific) against National Center for Biotechnology Information (NCBI) database with Chordata taxonomy (782,473 entries, downloaded in January 2017). Fragments and parents' tolerances were set at 0.80 Da and 5 ppm, respectively. The search parameters included trypsin as a protease with two allowed missed cleavages and carbamidomethylcysteine, methionine oxidation and acetylation of N-term protein as variable modifications. Mascot results obtained from the target and decoy databases searches were subjected to Scaffold 4.8.2 software (Proteome Software, Portland, OR), and displayed as clusters. Peptide and protein identification and validation were performed using the Peptide and Protein Prophet algorithms, respectively (95.0% probability, at least two different unique peptides), from three technical replicates. The abundance of identified proteins was estimated by calculating the emPAI using Scaffold Q+ software (version 4.4, Proteome Software).
      Keratins were not taken into consideration in the subsequent analysis as they might be either contaminant from human skin and/or chicken skin. The list of keratins identified in the analysis is however available (supplemental Data S2).

      Functional Annotation Using Gene Ontology

      Gene ontology terms annotations for biological processes and cellular components category provided by the GO consortium (http://www.geneontology.org/) were investigated using protein database with UniprotKB (http://www.uniprot.org), and genomic databases with Ensembl (http://www.ensembl.org) and NCBI (https://www.ncbi.nlm.nih.gov).

      Comparison Between Human and Chicken AF Proteomes

      IPI numbers and gene symbols corresponding to the proteins identified in the human AF were retrieved from the publication of Cho et al., (
      • Cho C.K.J.
      • Shan S.J.
      • Winsor E.J.
      • Diamandis E.P.
      Proteomics analysis of human amniotic fluid.
      ), which integrated all previous studies on the human AF proteome. Earlier versions of the human protein sequences were recovered at the European Bioinformatics Institute website (ftp://ftp.ebi.ac.uk/pub/databases/IPI/) and updated according to the Ensembl database. Human and chicken gene symbols were compared and for associated genes, protein sequences were aligned using BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi) to identify similarity and identity percentages between both species. Human orthologues were systematically checked using Ensembl compara (http://www.ensembl.org) to confirm previous alignments.

      Phylogenetic Analysis

      Phylogenetic branches of emergence were defined in order to determine the moment of appearance of each gene in the tree of life. Eight possible branches for gene birth were considered: Opisthokontas, (animals and fungi ∼ 1215 million years ago (MYA)), Bilateria, (bilateral animals ∼ 937 MYA), Chordata, (chordates ∼ 722 MYA), Vertebrata, (vertebrates ∼ 535 MYA), Tetrapoda, (tetrapods ∼ 371 MYA), Amniota, (amniotes ∼ 296 MYA), Sauropsida, (reptiles and birds ∼ 276 MYA), and Mammalia, (mammals ∼ 220 MYA). For all identified proteins, the corresponding Ensembl protein ID was retrieved from the Ensembl database and the related phylogenetic trees were analyzed to highlight the specificity of proteins evolution (http://www.ensembl.org) (
      • Vilella A.J.
      • Severin J.
      • Ureta-Vidal A.
      • Heng L.
      • Durbin R.
      • Birney E.
      EnsemblCompara GeneTrees: Complete, duplication-aware phylogenetic trees in vertebrates.
      ). Eighty-nine trees were studied. To complement the phylogenetic trees, the conservation of synteny was systematically observed using Genomicus (http://www.genomicus.biologie.ens.fr) and Mapviewer (https://www.ncbi.nlm.nih.gov/mapview) (
      • Tian X.
      • Gautron J.
      • Monget P.
      • Pascal G.
      What Makes an Egg Unique? Clues from Evolutionary Scenarios of Egg-Specific Genes.
      ,
      • Dufourny L.
      • Levasseur A.
      • Migaud M.
      • Callebaut I.
      • Pontarotti P.
      • Malpaux B.
      • Monget P.
      GPR50 is the mammalian ortholog of Mel1c: evidence of rapid evolution in mammals. BMC Evol.
      ). The gene was defined as conserved if both surrounding/adjacent genes were the same.

      Purification of Heparin Binding Proteins

      Heparin-Sepharose chromatography was performed according to manufacturer's instructions. Briefly, 2 ml of beads (Heparin Sepharose 6 Flast Flow, GE Healthcare, Sweden) were loaded onto a polypropylene column (Qiagen, Courtaboeuf, France) and washed with 10 volumes of water and 10 volumes of washing buffer (50 mm Tris-HCl, 150 mm NaCl, pH 7.4). After loading the crude sample (pool of ten individuals, five males and five females), the beads were washed extensively with the washing buffer until the absorbance at 220 nm reached zero, as previously reported (
      • Guyot N.
      • Labas V.
      • Harichaux G.
      • Chesse M.
      • Poirier J.C.
      • Nys Y.
      • Rehault-Godbert S.
      Proteomic analysis of egg white heparin-binding proteins: towards the identification of natural antibacterial molecules.
      ). Elution of bound proteins was achieved with 50 mm Tris-HCl, 2 m NaCl, pH 7.4, until the absorbance at 220 nm reached zero. Eluted fractions were desalted and concentrated by ultrafiltration (Ultracel-3K, Merck Millipore, Molsheim, France), and analyzed by 12.5% SDS-PAGE under nonreducing and nonboiling conditions to preserve protein integrity, followed by Coomassie Brilliant Blue G250 staining.

      Antibacterial Assays

      Antibacterial tests were conducted by direct detection of antibacterial activities after SDS-PAGE, a method adapted from Bhunia et al., (
      • Bhunia A.K.
      • Johnson M.C.
      • Ray B.
      Direct Detection of an antimicrobial peptide of Pediococcus-Acidilactici in sodium dodecyl sulfate polyacrylamide gel electrophoresis J.
      ). Pathogenic bacterial strains, Salmonella enterica, serovar Enteritidis ATCC 13076 (S.,E.) and Listeria monocytogenes, EGD strain (L.m,.) were provided by the International Centre for Microbial Resources (CIRM, https://www6.inra.fr/cirm_eng/Pathogenic-Bacteria) from the French National Institute for Agricultural Research (INRA, France). Precultures of S.,E. and L.m., were performed overnight in Trypticase Soy Broth (TSB, BD Biosciences, Le Pont de Claix, France) and in Brain Heart Infusion broth (BHI, BD Difco), respectively. This pre-culture was then used to inoculate a new culture broth (TSB or BHI) so that the midexponential phase was obtained after 3 or 4 h of incubation depending on strains, with shaking at 37 °C. Bacteria were centrifuged at 2000 × g, for 10 min at 4 °C, washed twice with cold 10 mm sodium phosphate buffer (pH 7.4), and resuspended in cold sodium phosphate buffer. Bacteria (7.5 × 106 Colony Forming Unit) were introduced in 25 ml of autoclaved nutrient-poor agar (10 mm phosphate buffer containing 0.03% TSB medium, 1% low-endosmosis agarose (w/v) (Sigma-Aldrich, Saint-Quentin-Fallavier, France), and 0.02% Tween 20.
      In parallel, maximum of 20 μg of protein pools (see above) were loaded on two identical gels and further separated by 15% SDS-PAGE under nonreducing and nonboiling conditions, while maintaining the electrophoresis system at 4 °C to avoid protein degradation. The first gel was stained with Coomassie Brilliant Blue G250 for further identification of antibacterial proteins by mass spectrometry. The second gel was dedicated to the antibacterial assay and was washed separately with 2.5% Triton X-100 and MilliQ water (4 × 15 min, 4 °C), to eliminate SDS and renature proteins. Thereafter, the washed gel was covered with the nutrient-poor agar containing bacteria and incubated for 3 h at 37 °C to allow protein diffusion in the agar. A second nutrient-rich agar (10 mm phosphate buffer containing 6% TSB medium, 1% low-endosmosis agarose (w/v)) was poured on the nutrient-poor agar to allow bacterial growth. After an overnight incubation at 37 °C, clear zones were defined as inhibition zones. These inhibition zones were superimposed on the Coomassie-stained gel to locate bands containing antibacterial proteins. These bands were cut from the Coomassie-stained gel and further processed as described above, for protein identification by mass spectrometry.
      Beta-microseminoprotein-like (BMSP) and avian beta-defensin 11 (AvBD11), two recently characterized antibacterial proteins from egg white were used as positive external controls (2 μg of proteins/well, data not shown). They were obtained as previously described (
      • Guyot N.
      • Labas V.
      • Harichaux G.
      • Chesse M.
      • Poirier J.C.
      • Nys Y.
      • Rehault-Godbert S.
      Proteomic analysis of egg white heparin-binding proteins: towards the identification of natural antibacterial molecules.
      ).

      Experimental Design and Statistical Rationale

      To address our scientific questions, we decided to use a conventional chicken laying strain (ISA Brown, Hendrix Genetics, St Brieuc, France) at 38 weeks of age, that were raised at the UE1295 Pôle d'Expe′rimentation Avicole de Tours (INRA, F-37380 Nouzilly, France). Eighty fertilized eggs were incubated under standard conditions after a three-day storage at 16 °C and 85% relative humidity to favor synchronization of developmental stages—knowing that conditions and duration of storage prior to incubation negatively impact embryo survival and development. The developmental stages were confirmed using the atlas of chicken developmental stages (
      • Hamburger V.
      • Hamilton H.L.
      A series of normal stages in the development of the chick embryo.
      ). Egg weight and eggshell quality were both checked and protein profiles of each individual sample were analyzed by SDS-PAGE (supplemental Data S1, sheets #1 and #2). In parallel, the sex of the embryo was determined for all corresponding samples as described under Experimental Procedures to generate a pool of combined male and females AF that could encompass most proteins composing AF, regardless of the sex. The combination of all these parameters allowed us to define 12 samples with an equilibrated sex ratio that were all homogenous in terms of stages of development, biochemical parameters (pH, osmolality, absorbance spectrum, etc.), and SDS-PAGE protein profiles. All experiments were conducted in compliance with the European legislation on the “Protection of Animals Used for Experimental and Other Scientific Purposes” (2010/63/UE) and under the supervision of an authorized scientist (S. Réhault-Godbert, Authorization no. 37–144). Two complementary bottom-up proteomic strategies (GeLC-MS/MS and shotgun analyses) were combined to give a representative overview of protein diversity in chicken AF samples. Protein sequences were retrieved from Scaffold and blasted to check their GenBank accession numbers (NCBI), and to find corresponding Ensembl and Uniprot IDs. Chicken proteins identified in other species were also searched by BLAST alignments. Functional annotation and search for antibacterial candidates were performed by combining Ensembl, Uniprot and NCBI data on chicken proteins and their orthologues. The identification of antibacterial candidates was completed by investigating protein and peptide antibacterial domains using specific sequence analyzing tools such as InterPro (https://www.ebi.ac.uk/interpro/). To produce enriched fractions, we defined optimal conditions (volume of samples, volume of heparin-Sepharose beads) depending on the initial concentration of proteins in AF and egg white samples from ED0, ED8, ED11, ED14 or ED16 stages. All samples (raw, flow-through and eluted fractions) were analyzed by SDS-PAGE. Protein profiles of egg white at ED11 and ED16 were compared with previously published data for experimental validation (
      • Guyot N.
      • Labas V.
      • Harichaux G.
      • Chesse M.
      • Poirier J.C.
      • Nys Y.
      • Rehault-Godbert S.
      Proteomic analysis of egg white heparin-binding proteins: towards the identification of natural antibacterial molecules.
      ,
      • Guyot N.
      • Rehault-Godbert S.
      • Slugocki C.
      • Harichaux G.
      • Labas V.
      • Helloin E.
      • Nys Y.
      Characterization of egg white antibacterial properties during the first half of incubation: A comparative study between embryonated and unfertilized eggs.
      ). For antibacterial assays, at least three independent assays were performed on Listeria monocytogenes, and Salmonella enterica, Enteritidis strains, using two positive controls (one peptide and one protein) purified from chicken egg white. The phylogenetic analysis and corresponding figures were conducted combining data available in Ensembl databases and in literature.

      DISCUSSION

      In amniotes, AF plays a crucial role in maintaining a stable and protective milieu. In humans, the exhaustive characterization of its biochemical components (including proteins) at various stages of gestation in normal and pathological situations constitutes an extremely valuable approach to help in the diagnosis of human fetal disorders and infections. In avian species, there is evidence that AF also participates in homeostasis around the embryo (
      • Da Silva M.
      • Labas V.
      • Nys Y.
      • Rehault-Godbert S.
      Investigating proteins and proteases composing amniotic and allantoic fluids during chicken embryonic development.
      ) and in its protection against physical constraints (
      • Romanoff A.L.
      ). Nevertheless, little is known about the physiological role of AF proteins in birds, and there is to date little proteomic data available on this fluid for avian species. Yet, we suspect that the development of the embryo in an egg, with no mother-connected tissues, requires some inherent specificities and structural particularities that may influence the AF protein profile and composition in birds.
      In the present work, we identified 91 nonredundant proteins with high confidence, in the chicken AF at ED11, before egg white transfer, which is higher than the 47 proteins identified in a preliminary study (
      • Da Silva M.
      • Labas V.
      • Nys Y.
      • Rehault-Godbert S.
      Investigating proteins and proteases composing amniotic and allantoic fluids during chicken embryonic development.
      ). This number is however far less than the number of proteins identified in two surveys performed on human AF (219 (
      • Michaels J.E.A.
      • Dasari S.
      • Pereira L.
      • Reddy A.P.
      • Lapidus J.A.
      • Lu X.F.
      • Jacob T.
      • Thomas A.
      • Rodland M.
      • Roberts C.T.
      • Gravett M.G.
      • Nagalla S.R.
      Comprehensive proteomic analysis of the human amniotic fluid proteome: Gestational age-dependent changes.
      ) and 923 (
      • Cho C.K.J.
      • Shan S.J.
      • Winsor E.J.
      • Diamandis E.P.
      Proteomics analysis of human amniotic fluid.
      )). This difference can be partly explained by the various exchanges with maternal tissues that take place during gestation in humans, which result in some specific AF protein profiles, and the fact that in humans, metabolic wastes originating from the embryo are indeed recovered in the AF, which is not the case in the chicken model (
      • Underwood M.A.
      • Gilbert W.M.
      • Sherman M.P.
      Amniotic fluid: not just fetal urine anymore.
      ).
      All combined results indicate that more than half of the proteins identified in the chicken AF at ED11 have orthologues that have been identified as components of the human AF, which highlights that the overall protein composition of the AF before egg white transfer exhibits high similarities with the human AF. This is strengthened by the observation that, five of the top-ten high abundant proteins identified in the chicken AF at ED11 (Table I - ALB, TF, AFP, GC, and APOA1) are also listed in the 15 high abundant proteins recovered in human AF (
      • Michaels J.E.A.
      • Dasari S.
      • Pereira L.
      • Reddy A.P.
      • Lapidus J.A.
      • Lu X.F.
      • Jacob T.
      • Thomas A.
      • Rodland M.
      • Roberts C.T.
      • Gravett M.G.
      • Nagalla S.R.
      Comprehensive proteomic analysis of the human amniotic fluid proteome: Gestational age-dependent changes.
      ,
      • Cho C.K.J.
      • Shan S.J.
      • Winsor E.J.
      • Diamandis E.P.
      Proteomics analysis of human amniotic fluid.
      ). It is notable that the TF identified in the chicken AF corresponds to ovotransferrin, which shares 51.76% and 51.73% sequence identity with human serotransferrin (P02787) and human lactotransferrin (P02788), respectively. This moderate sequence identity between ovotransferrin and its human homologs, together with some specific structural features, such as glycosylation and number of disulfide bridges (
      • Metz-Boutigue M.H.
      • Jolles J.
      • Mazurier J.
      • Schoentgen F.
      • Legrand D.
      • Spik G.
      • Montreuil J.
      • Jolles P.
      Human lactotransferrin: amino acid sequence and structural comparisons with other transferrins.
      ), suggest similar functions with regard to their iron-binding capacity and storage, but also possibly diverging functions. As an example, both lactotransferrin and ovotransferrin, but not serotransferrin, are annotated as antibacterial proteins (
      • Bellamy W.
      • Takase M.
      • Yamauchi K.
      • Wakabayashi H.
      • Kawase K.
      • Tomita M.
      Identification of the bactericidal domain of lactoferrin.
      ,
      • Baron F.
      • Jan S.
      • Gonnet F.
      • Pasco M.
      • Jardin J.
      • Giudici B.
      • Gautier M.
      • Guerin-Dubiard C.
      • Nau F.
      Ovotransferrin plays a major role in the strong bactericidal effect of egg white against the Bacillus cereus group.
      ). Interestingly, two of the top-ten abundant proteins have no assigned functions: OVAL and SPINK7. These two proteins are major proteins of egg white (
      • Mann K.
      The chicken egg white proteome.
      ) and appear belatedly in the timeline of evolution, with OVAL and very likely SPINK7, being specific to birds (Fig. 9).
      Because of these similitudes in protein composition, most functions attributed to the most abundant proteins composing the human AF resemble that of the chicken AF: (1) metabolism and transport of vitamins, lipids and hormones, (2) immune response, and (3) hemostasis and homeostasis. The analysis of the list of chicken AF proteins using automatic tools dedicated to Gene Ontology annotation, emphasized functions associated with many aspects of developmental biology such as morphogenesis and organogenesis, which include cell proliferation, adhesion and migration processes (Fig. 5). These functions were also significantly highlighted in the analysis of human AF proteins (
      • Michaels J.E.A.
      • Dasari S.
      • Pereira L.
      • Reddy A.P.
      • Lapidus J.A.
      • Lu X.F.
      • Jacob T.
      • Thomas A.
      • Rodland M.
      • Roberts C.T.
      • Gravett M.G.
      • Nagalla S.R.
      Comprehensive proteomic analysis of the human amniotic fluid proteome: Gestational age-dependent changes.
      ,
      • Cho C.K.J.
      • Shan S.J.
      • Winsor E.J.
      • Diamandis E.P.
      Proteomics analysis of human amniotic fluid.
      ). However, the biological significance of these functions in the chicken and human AF are likely to be overestimated since most proteins annotated with this function are very-low-abundance proteins and may reflect some embryonic or extraembryonic tissues desquamation (Supplemental data S2 and S3, sheet #2).
      Overall, it is noteworthy that of the ten most abundant proteins in the chicken AF, four of them are major egg white proteins (OVAL, SPINK7, LYZ, TF) (
      • Mann K.
      The chicken egg white proteome.
      ) whereas ALB, APOA1, GC, TTR, APOC3 are egg yolk abundant proteins (
      • Mann K.
      • Mann M.
      The chicken egg yolk plasma and granule proteomes.
      ). Gene expression of OVAL, SPINK7, LYZ and TF, which together account for about 80% of the egg white proteins, is under hormonal control in the oviduct and will not be expressed until the onset of sexual maturity in hens (
      • Palmiter R.D.
      Regulation of protein synthesis in chick oviduct. I. Independent regulation of ovalbumin, conalbumin, ovomucoid, and lysozyme induction.
      ). OVAL, SPINK7 and TF are thus likely to flow from egg white into the amniotic sac, rather than being expressed by embryonic tissues. As for LYZ, the ongoing hypothesis is that it also originates from egg white although it is also expressed by a wide range of tissues (
      • Nakano T.
      • Graf T.
      Goose-type lysozyme gene of the chicken: sequence, genomic organization and expression reveals major differences to chicken-type lysozyme gene.
      ). On the other hand, APOA1, ALB, GC, TTR and APOC3 represent high-abundance proteins of egg yolk (
      • Mann K.
      • Mann M.
      The chicken egg yolk plasma and granule proteomes.
      ), which also suggests that they are somehow transferred to the chicken AF from the egg yolk, or from the yolk sac where they are highly expressed (
      • Yadgary L.
      • Wong E.A.
      • Uni Z.
      Temporal transcriptome analysis of the chicken embryo yolk sac.
      ). The molecular mechanisms by which egg white and egg yolk proteins enter the AF during the first half of incubation are still not completely understood (
      • Sugimoto Y.
      • Sanuki S.
      • Ohsako S.
      • Higashimoto Y.
      • Kondo M.
      • Kurawaki J.
      • Ibrahim H.R.
      • Aoki T.
      • Kusakabe T.
      • Koga K.
      Ovalbumin in developing chicken eggs migrates from egg white to embryonic organs while changing its conformation and thermal stability.
      ,
      • Da Silva M.
      • Labas V.
      • Nys Y.
      • Rehault-Godbert S.
      Investigating proteins and proteases composing amniotic and allantoic fluids during chicken embryonic development.
      ). In contrast, the embryonic origin of AFP is unequivocal since it is a fetus-specific protein present in a range of (extra)embryonic tissues, highly expressed in the yolk sac and in the embryonic liver (
      • Slade B.
      • Milne J.
      Localization and synthesis of alpha-fetoprotein in the chicken.
      ). Although all these proteins are the most abundant proteins identified in the chicken AF at ED11, their protein concentration in the AF at this stage (0.01 mg/ml) is not comparable to those recovered in the egg yolk (>10 mg/ml, 22% of dry matter (
      • Anton M.
      Egg yolk: structures, functionalities and processes.
      )) and egg white (380 mg/ml (
      • Guyot N.
      • Rehault-Godbert S.
      • Slugocki C.
      • Harichaux G.
      • Labas V.
      • Helloin E.
      • Nys Y.
      Characterization of egg white antibacterial properties during the first half of incubation: A comparative study between embryonated and unfertilized eggs.
      )). The low protein concentration of chicken AF at ED11 suggests that the presence of proteins in this fluid may reflect some passive transfer from these egg compartments toward AF, rather than an active mechanism. This concentration is also lower as compared with that of human AF: it is about 4 mg/ml at 15 weeks of gestation (
      • Romero R.
      • Kusanovic J.P.
      • Gotsch F.
      • Erez O.
      • Vaisbuch E.
      • Mazaki-Tovi S.
      • Moser A.
      • Tam S.
      • Leszyk J.
      • Master S.R.
      • Juhasz P.
      • Pacora P.
      • Ogge G.
      • Gomez R.
      • Yoon B.H.
      • Yeo L.
      • Hassan S.S.
      • Rogers W.T.
      Isobaric labeling and tandem mass spectrometry: A novel approach for profiling and quantifying proteins differentially expressed in amniotic fluid in preterm labor with and without intra-amniotic infection/inflammation.
      ), it reaches its maximum (6 mg/ml) at 22–27 weeks of gestation and starts to decrease to about 2 mg/ml at 37–40 weeks of gestation (
      • Malik G.K.
      • Sinha S.M.
      • Saksena P.N.
      • Kapoor A.K.
      • Mehra P.
      • Bagchi M.
      • Agarwal D.K.
      • Tuteja N.
      Amniotic fluid proteins in relation to fetal maturity. Indian J.
      ). It also highlights that chicken AF cannot meet embryo's requirements in energy up to ED11, suggesting that during the first half of incubation, the egg yolk is the major source of energy/lipoproteins for the embryo. Some proteolytic activities (
      • Da Silva M.
      • Labas V.
      • Nys Y.
      • Rehault-Godbert S.
      Investigating proteins and proteases composing amniotic and allantoic fluids during chicken embryonic development.
      ) have been however detected in AF at ED11, but, because of the low abundance of the corresponding proteins, the biological significance of these activities in AF remains questionable as compared with the egg yolk, where hydrolytic enzymes are concentrated (
      • Mann K.
      • Mann M.
      The chicken egg yolk plasma and granule proteomes.
      ,
      • Retzek H.
      • Steyrer E.
      • Sanders E.J.
      • Nimpf J.
      • Schneider W.J.
      Molecular cloning and functional characterization of chicken cathepsin D, a key enzyme for yolk formation.
      ). The same observation can be made for the faint antibacterial activities detected at ED11 in chicken AF, compared with egg white, which concentrates high amounts of antibacterial proteins such as active lysozyme (
      • Guyot N.
      • Rehault-Godbert S.
      • Slugocki C.
      • Harichaux G.
      • Labas V.
      • Helloin E.
      • Nys Y.
      Characterization of egg white antibacterial properties during the first half of incubation: A comparative study between embryonated and unfertilized eggs.
      ,
      • Mann K.
      The chicken egg white proteome.
      ).
      These considerations along with the detection of antibacterial activities, corroborates the role of chicken AF in protecting the embryo against a range of potential physical, physicochemical and antibacterial threats. Moreover, it also emphasizes some particularities such as the presence in this fluid of bird-specific proteins involved in embryonic nutrition, eggshell biomineralization and egg defense (OVAL, OVALY, OVALX, OC-116, AvBD11) and likely SPINK7/ovomucoid and SPINK5/ovoinhibitor, recently renamed SPIK7 and SPIK5 in the NCBI Gene database (May 2017) and whose functions are still speculative. We also identified some oviparous-specific proteins (VTG1, VTG2, AVD, APOV1) (Fig. 9B,), that appear concomitantly with the appearance of the egg-laying type of reproduction. Additionally, this analysis revealed the presence of 43 proteins that have been uniquely found in the chicken AF as compared with human AF (Fig. 6). Nevertheless, the high number of proteins reported for human AF as compared with chicken AF, merits further studies. A comprehensive study of the proteome profiling of the allantoic fluid would probably answer some of these questions, as we might find allantoic fluid-specific proteins whose orthologues have been identified in human AF. Future studies of this specific fluid would confirm or not whether proteins from both chicken amniotic and allantoic fluids together encompass most proteins identified in the human AF.
      To conclude, the story of the chicken AF remains quite simple up to ED11, with a protein composition that is comparable to that of human AF or at least with similar general functions. However, the protein composition of AF is deeply revised after egg white transfer, which occurs between ED11 and ED12 (
      • Da Silva M.
      • Labas V.
      • Nys Y.
      • Rehault-Godbert S.
      Investigating proteins and proteases composing amniotic and allantoic fluids during chicken embryonic development.
      ). The AF protein profile then completely merges with the egg white protein profile (Fig. 3A,) (
      • Sugimoto Y.
      • Sanuki S.
      • Ohsako S.
      • Higashimoto Y.
      • Kondo M.
      • Kurawaki J.
      • Ibrahim H.R.
      • Aoki T.
      • Kusakabe T.
      • Koga K.
      Ovalbumin in developing chicken eggs migrates from egg white to embryonic organs while changing its conformation and thermal stability.
      ,
      • Da Silva M.
      • Labas V.
      • Nys Y.
      • Rehault-Godbert S.
      Investigating proteins and proteases composing amniotic and allantoic fluids during chicken embryonic development.
      ,
      • Nelson T.C.
      • Groth K.D.
      • Sotherland P.R.
      Maternal investment and nutrient use affect phenotype of American alligator and domestic chicken hatchlings.
      ), with ten major proteins consisting of OVAL, LYZ, TF, OVALY, SPINK7, VMO1, SPINK5, AVD, OVALX, and CST3 (
      • Mann K.
      The chicken egg white proteome.
      ). Despite the presence of AF hydrolytic enzymes (
      • Da Silva M.
      • Labas V.
      • Nys Y.
      • Rehault-Godbert S.
      Investigating proteins and proteases composing amniotic and allantoic fluids during chicken embryonic development.
      ), no major proteolytic degradation of egg white proteins was detected after its inflow into the amniotic cavity, up to ED19. These data suggest that abundant egg white antiproteases, namely SPINK7, SPINK5, cystatin (CST3) and ovostatin (OVST), remain active during the entire duration of incubation. It is rather interesting to note that the inflow of egg white reinforces some proteins that were already present as major proteins in ED11-AF (OVAL, LYZ, TF and SPINK7, Table I). More than half of these major egg white proteins are related to innate defense (LYZ, TF, VMO1, SPINK5, AVD, OVALX) while three of them are protease inhibitors (SPINK5, SPINK7, CST3). Therefore, we suspected that chicken AF enriched in egg white proteins from ED12 onwards, acquires an increased antibacterial potential. An in-gel antibacterial assay was developed to better appreciate the relevance and effective activity of these so-called antibacterial proteins in the AF-egg white mixture. We showed that ED16-AF displays higher antibacterial potential against the two bacterial strains tested, namely Listeria monocytogenes, (Gram-positive strain), and Salmonella enterica, Enteritidis (Gram-negative strain) compared with ED11-AF. A total of 29 proteins were identified in the corresponding zones lacking bacterial growth (Table II), including LYZ, AvBD11, VMO1, AVD, LOC101750704 (BMSP), OVALX, TF, OC-17, BPIFB2 and MDK that were all previously reported to exhibit antibacterial activities (
      • Guyot N.
      • Labas V.
      • Harichaux G.
      • Chesse M.
      • Poirier J.C.
      • Nys Y.
      • Rehault-Godbert S.
      Proteomic analysis of egg white heparin-binding proteins: towards the identification of natural antibacterial molecules.
      ,
      • Wellman-Labadie O.
      • Lakshminarayanan R.
      • Hincke M.T.
      Antimicrobial properties of avian eggshell-specific C-type lectin-like proteins.
      ,
      • Rehault-Godbert S.
      • Labas V.
      • Helloin E.
      • Herve-Grepinet V.
      • Slugocki C.
      • Berges M.
      • Bourin M.-C.
      • Brionne A.
      • Poirier J.-C.
      • Gautron J.
      • Coste F.
      • Nys Y.
      Ovalbumin-related protein X is a heparin-binding Ov-serpin exhibiting antimicrobial activities.
      ,
      • Svensson S.L.
      • Pasupuleti M.
      • Walse B.
      • Malmsten M.
      • Morgelin M.
      • Sjogren C.
      • Olin A.I.
      • Collin M.
      • Schmidtchen A.
      • Palmer R.
      • Egesten A.
      Midkine and pleiotrophin have bactericidal properties: preserved antibacterial activity in a family of heparin-binding growth factors during evolution.
      ,
      • Baron F.
      • Jan S.
      • Gonnet F.
      • Pasco M.
      • Jardin J.
      • Giudici B.
      • Gautier M.
      • Guerin-Dubiard C.
      • Nau F.
      Ovotransferrin plays a major role in the strong bactericidal effect of egg white against the Bacillus cereus group.
      ,
      • Maehashi K.
      • Ueda M.
      • Matano M.
      • Takeuchi J.
      • Uchino M.
      • Kashiwagi Y.
      • Watanabe T.
      Biochemical and functional characterization of transiently expressed in neural precursor (TENP) protein in emu egg white.
      ,
      • Elo H.A.
      • Korpela J.
      The occurrence and production of avidin: a new conception of the high-affinity biotin-binding protein.
      ,
      • Matulova M.
      • Rajova J.
      • Vlasatikova L.
      • Volf J.
      • Stepanova H.
      • Havlickova H.
      • Sisak F.
      • Rychlik I.
      Characterization of chicken spleen transcriptome after infection with Salmonella enterica serovar Enteritidis.
      ,
      • Nakimbugwe D.
      • Massehalck B.
      • Atanassova M.
      • Zewdie-Bosuner A.
      • Michiels C.W.
      Comparison of bactericidal activity of six lysozymes at atmospheric pressure and under high hydrostatic pressure.
      ,
      • Sellier N.
      • Vidal M.L.
      • Baron F.
      • Michel J.
      • Gautron J.
      • Protais M.
      • Beaumont C.
      • Gautier M.
      • Nys Y.
      Estimations of repeatability and heritability of egg albumen antimicrobial activity and of lysozyme and ovotransferrin concentrations.
      ,
      • Herve-Grepinet V.
      • Rehault-Godbert S.
      • Labas V.
      • Magallon T.
      • Derache C.
      • Lavergne M.
      • Gautron J.
      • Lalmanach A.C.
      • Nys Y.
      Purification and Characterization of Avian beta-Defensin 11, an Antimicrobial Peptide of the Hen Egg.
      ). These results demonstrate that the antibacterial potential of egg white proteins is not impaired by the incubation temperature, nor by proteolytic degradation (thanks to egg white antiproteases), and that its antimicrobial potential remains effective after transfer into the AF. However, subtle differences are detected in AF at ED16 after completion of egg white transfer, but also in remnant egg white at ED8–14. Indeed, additional antibacterial zones were visualized on anti-Listeria monocytogenes, assays performed with ED16-AF and ED8-14-EW (Fig. 7., Fig. 8., respectively). Unexpectedly, they correspond to proteins or protein complexes/associations of very high molecular mass (>250 kDa). These high molecular mass complexes were identified as a mixture of egg white proteins: clusterin (CLU, 62 kDa), SPINK5 (52 kDa), OVALX (44 kDa), AVD (tetramer of 17 kDa units), OVAL (43 kDa), TF (78 kDa), ovomucin (234 kDa), LYZ (14 kDa), BPIFB2 (47 kDa), etc. The progressive appearance of these complexes suggests some specific regulation occurring during incubation. Indeed, the re-distribution of the egg white water toward extraembryonic sacs during the first half of the development, concentrates egg white proteins, thus promoting protein-protein interactions between antibacterial proteins/peptides of low molecular masses (LYZ, LOC101750704 (BMSP), AvBD11, etc.) and proteins of higher molecular masses (ovomucin). These protein complexes do not seem to affect the antibacterial protein/peptide activities, which are still effective even before oral absorption by the embryo (Fig. 8), thus forming another barrier against bacteria around the embryo's body. As the embryo moves and develops in the AF until hatching, it can be hypothesized that this mixture of antimicrobial molecules and mucins may also constitute a protective biofilm deposited on the embryonic feathers and skin to protect the embryo and the newborn chick. This proposal is partly supported by the presence of lysozyme and defensin-like molecules in human vernix caseoa, (
      • Tollin M.
      • Bergsson G.
      • Kai-Larsen Y.
      • Lengqvist J.
      • Sjovall J.
      • Griffiths W.
      • Skuladottir G.V.
      • Haraldsson A.
      • Jornvall H.
      • Gudmundsson G.H.
      • Agerberth B.
      Vernix caseosa as a multi-component defence system based on polypeptides, lipids and their interactions.
      ,
      • Yoshio H.
      • Tollin M.
      • Gudmundsson G.H.
      • Lagercrantz H.
      • Jornvall H.
      • Marchini G.
      • Agerberth B.
      Antimicrobial polypeptides of human vernix caseosa and amniotic fluid: implications for newborn innate defense.
      ), a protective lipid-rich substance that covers the skin of the human fetus during the last trimester of gestation and the newborn. Whether this enhanced antimicrobial potential of AF prior to its swallowing has some relevant biological significance with respect to the protection of the embryonic digestive tract is also of interest. Indeed, the fate of the AF-egg white protein mixture that is ingested by the embryo is still poorly understood. These proteins remain very stable in egg white during incubation but also in the AF (
      • Sugimoto Y.
      • Sanuki S.
      • Ohsako S.
      • Higashimoto Y.
      • Kondo M.
      • Kurawaki J.
      • Ibrahim H.R.
      • Aoki T.
      • Kusakabe T.
      • Koga K.
      Ovalbumin in developing chicken eggs migrates from egg white to embryonic organs while changing its conformation and thermal stability.
      ,
      • Da Silva M.
      • Labas V.
      • Nys Y.
      • Rehault-Godbert S.
      Investigating proteins and proteases composing amniotic and allantoic fluids during chicken embryonic development.
      ) and all along the length of the digestive tract of the embryo, up to ED19 (
      • Nelson T.C.
      • Groth K.D.
      • Sotherland P.R.
      Maternal investment and nutrient use affect phenotype of American alligator and domestic chicken hatchlings.
      ). Similarly, some egg white typical profiles can be visualized in the yolk sac contents at ED20 just before emergence of the chick, with OVAL, LYZ and TF as major proteins (
      • Geelhoed S.E.
      • Conklin J.L.
      An electrophoretic study of proteins in chick embryonic fluids.
      ,
      • Nelson T.C.
      • Groth K.D.
      • Sotherland P.R.
      Maternal investment and nutrient use affect phenotype of American alligator and domestic chicken hatchlings.
      ). These data together with the fact that OVAL was recovered as a native form in the central nervous system and in other embryonic organs (
      • Sugimoto Y.
      • Sanuki S.
      • Ohsako S.
      • Higashimoto Y.
      • Kondo M.
      • Kurawaki J.
      • Ibrahim H.R.
      • Aoki T.
      • Kusakabe T.
      • Koga K.
      Ovalbumin in developing chicken eggs migrates from egg white to embryonic organs while changing its conformation and thermal stability.
      ), suggest that this protein and possibly related proteins OVALX and OVALY may have other functions than nutrition (
      • Da Silva M.
      • Beauclercq S.
      • Harichaux G.
      • Labas V.
      • Guyot N.
      • Gautron J.
      • Nys Y.
      • Rehault-Godbert S.
      The family secrets of avian egg-specific ovalbumin and its related proteins Y and X.
      ). From these data, we infer that intrinsic egg yolk proteases exhibit limited proteolytic activities within the egg white contents at this stage. This can be partly explained by the gradual increase of the yolk pH, which impacts the activity of aspartic proteases (
      • Cunningham M.
      • Tang J.
      Purification and properties of cathepsin D from porcine spleen.
      ), such as chicken cathepsin D, a key enzyme in yolk processing (
      • Retzek H.
      • Steyrer E.
      • Sanders E.J.
      • Nimpf J.
      • Schneider W.J.
      Molecular cloning and functional characterization of chicken cathepsin D, a key enzyme for yolk formation.
      ). It seems that everything converges to protect egg white-AF protein content from uncontrolled proteolytic and thermal degradation (presence of numerous proteases inhibitors together with the progressive increase in thermostable S-ovalbumin during incubation (
      • Castellano A.C.
      • Barteri M.
      • Bianconi A.
      • Bruni F.
      • Della Longa S.
      • Paolinelli C.
      Conformational changes involved in the switch from ovalbumin to S-ovalbumin.
      ,
      • Yamasaki M.
      • Takahashi N.
      • Hirose M.
      Crystal structure of S-ovalbumin as a non-loop-inserted thermostabilized serpin form.
      )), to be utilized by the embryo and/or the chick at a very precise moment of its development/growth.
      Besides its major interest for comparative biologic approaches and for deciphering the functions and specificities of the avian extraembryonic compartments, these results (protein profile and composition) constitute a reference starting point for analyses of pathological conditions due to infection or impaired development occurring during the chicken embryonic development. As examples, in humans, differences in concentration of AFP, defensins, vitamin-binding proteins, APOA1, and TTR in AF (also identified in chicken AF) have been associated with many metabolic and developmental disorders such as trisomy, developmental delays, preterm-labor, inflammation or infections (
      • Spencer K.
      • Muller F.
      • Aitken D.A.
      Biochemical markers of trisomy 21 in amniotic fluid.
      ,
      • Liu Y.
      • Liu Y.
      • Du C.
      • Zhang R.
      • Feng Z.
      • Zhang J.
      Diagnostic value of amniotic fluid inflammatory biomarkers for subclinical chorioamnionitis.
      ,
      • Hsu T.Y.
      • Lin H.
      • Hung H.N.
      • Yang K.D.
      • Ou C.Y.
      • Tsai C.C.
      • Cheng H.H.
      • Chung S.H.
      • Cheng B.H.
      • Wong Y.H.
      • Chou A.K.
      • Hsiao C.C.
      Two-Dimensional differential gel electrophoresis to identify protein biomarkers in amniotic fluid of Edwards Syndrome (Trisomy 18) pregnancies.
      ). These data combined with other high throughput methods such as metabolomics or epigenetics, and general egg quality traits, may constitute useful tools for poultry production. Indeed, these approaches may help to further investigate the impact of chicken lines, housing systems and nutrition of hens (
      • Wolanski N.J.
      • Renema R.A.
      • Robinson F.E.
      • Carney V.L.
      • Fancher B.I.
      Relationships among egg characteristics, chick measurements, and early growth traits in ten broiler breeder strains.
      ,
      • Karcher D.M.
      • Jones D.R.
      • Abdo Z.
      • Zhao Y.
      • Shepherd T.A.
      • Xin H.
      Impact of commercial housing systems and nutrient and energy intake on laying hen performance and egg quality parameters.
      ) or of more subtle egg manipulations including conditions of egg storage prior to the incubation (
      • Bakst M.R.
      • Welch G.R.
      • Fetterer R.
      • Miska K.
      Impact of broiler egg storage on the relative expression of selected blastoderm genes associated with apoptosis, oxidative stress, and fatty acid metabolism.
      ) or thermal manipulation of eggs (
      • Loyau T.
      • Hennequet-Antier C.
      • Coustham V.
      • Berri C.
      • Leduc M.
      • Crochet S.
      • Sannier M.
      • Duclos M.J.
      • Mignon-Grasteau S.
      • Tesseraud S.
      • Brionne A.
      • Metayer-Coustard S.
      • Moroldo M.
      • Lecardonnel J.
      • Martin P.
      • Lagarrigue S.
      • Yahav S.
      • Collin A.
      Thermal manipulation of the chicken embryo triggers differential gene expression in response to a later heat challenge.
      ), on many aspects of chicken embryo development and health. We believe that the identification of a set of molecular markers for abnormal embryonic development in chicken extraembryonic fluids will contribute to the development of tools to improve poultry managements and, to increase the robustness of chicks and chickens to help them face contrasted and changing environments.

      DATA AVAILABILITY

      Data are available via, the PRIDE partner repository with the data set identifiers PXD008046 and 10.6019/PXD008046.

      Acknowledgments

      We thank Joël Delaveau and Christophe Rat (INRA, UE PEAT 609, F-37380 Nouzilly, France) for providing fertilized eggs; Angelina Trotereau and Nathalie Winter (UMR UR1282 Infectiologie Animale et Santé Publique, F-37380 Nouzilly, France) who gave us access to L2 laboratories, which were required to perform antimicrobial assays using bacterial pathogenic strains.

      REFERENCES

        • Moran Jr, E.T.
        Nutrition of the developing embryo and hatchling.
        Poult. Sci. 2007; 86: 1043-1049
        • Bellairs R.
        • Osmond M.
        The Atlas of Chick Development. Third edition Ed. Academic Press, Oxford, UK2014
        • Sheng G.
        • Foley A.C.
        Diversification and conservation of the extraembryonic tissues in mediating nutrient uptake during amniote development.
        Ann. N.Y. Acad. Sci. 2012; 1271: 97-103
        • Yadgary L.
        • Wong E.A.
        • Uni Z.
        Temporal transcriptome analysis of the chicken embryo yolk sac.
        BMC Genomics. 2014; 15: 690
        • Gabrielli M.G.
        • Accili D.
        The chick chorioallantoic membrane: a model of molecular, structural, and functional adaptation to transepithelial ion transport and barrier function during embryonic development.
        J. Biomed. Biotechnol. 2010; 2010: 940741
        • Underwood M.A.
        • Gilbert W.M.
        • Sherman M.P.
        Amniotic fluid: not just fetal urine anymore.
        J. Perinatol. 2005; 25: 341-348
        • Bakalar N.
        Sensory science: partners in flavour.
        Nature. 2012; 486: S4-S5
        • Orczyk-Pawilowicz M.
        • Jawien E.
        • Deja S.
        • Hirnle L.
        • Zabek A.
        • Mlynarz P.
        Metabolomics of human amniotic fluid and maternal plasma during normal pregnancy.
        PLoS ONE. 2016; 11: e0152740
        • Michaels J.E.A.
        • Dasari S.
        • Pereira L.
        • Reddy A.P.
        • Lapidus J.A.
        • Lu X.F.
        • Jacob T.
        • Thomas A.
        • Rodland M.
        • Roberts C.T.
        • Gravett M.G.
        • Nagalla S.R.
        Comprehensive proteomic analysis of the human amniotic fluid proteome: Gestational age-dependent changes.
        J. Proteome Res. 2007; 6: 1277-1285
        • Romero R.
        • Kusanovic J.P.
        • Gotsch F.
        • Erez O.
        • Vaisbuch E.
        • Mazaki-Tovi S.
        • Moser A.
        • Tam S.
        • Leszyk J.
        • Master S.R.
        • Juhasz P.
        • Pacora P.
        • Ogge G.
        • Gomez R.
        • Yoon B.H.
        • Yeo L.
        • Hassan S.S.
        • Rogers W.T.
        Isobaric labeling and tandem mass spectrometry: A novel approach for profiling and quantifying proteins differentially expressed in amniotic fluid in preterm labor with and without intra-amniotic infection/inflammation.
        J. Matern. Fetal Neonatal. Med. 2010; 23: 261-280
        • Cho C.K.J.
        • Shan S.J.
        • Winsor E.J.
        • Diamandis E.P.
        Proteomics analysis of human amniotic fluid.
        Mol. Cell. Proteomics. 2007; 6: 1406-1415
        • Yoshizaki N.
        • Ito Y.
        • Hori H.
        • Saito H.
        • Iwasawa A.
        Absorption, transportation and digestion of egg white in quail embryos.
        Dev. Growth Differ. 2002; 44: 11-22
        • Geelhoed S.E.
        • Conklin J.L.
        An electrophoretic study of proteins in chick embryonic fluids.
        J. Exp. Zool. 1966; 162: 257-261
        • Baintner K.
        • Fehér G.
        Fate of egg white trypsin inhibitor and start of proteolysis in developing chick embryo and newly hatched chick.
        Dev. Biol. 1974; 36: 272-278
        • Cirkvenčič N.
        • Narat M.
        • Dovč P.
        • Benčina D.
        Distribution of chicken cathepsins B and L, cystatin and ovalbumin in extra-embryonic fluids during embryogenesis.
        Br. Poult. Sci. 2012; 53: 623-630
        • Sugimoto Y.
        • Sanuki S.
        • Ohsako S.
        • Higashimoto Y.
        • Kondo M.
        • Kurawaki J.
        • Ibrahim H.R.
        • Aoki T.
        • Kusakabe T.
        • Koga K.
        Ovalbumin in developing chicken eggs migrates from egg white to embryonic organs while changing its conformation and thermal stability.
        J. Biol. Chem. 1999; 274: 11030-11037
        • Muramatsu T.
        • Hiramoto K.
        • Koshi N.
        • Okumura J.
        • Miyoshi S.
        • Mitsumoto T.
        Importance of albumen content in whole-body protein synthesis of the chicken embryo during incubation.
        Br. Poult. Sci. 1990; 31: 101-106
        • Da Silva M.
        • Labas V.
        • Nys Y.
        • Rehault-Godbert S.
        Investigating proteins and proteases composing amniotic and allantoic fluids during chicken embryonic development.
        Poult. Sci. 2017; 96: 2931-2941
        • Romanoff A.L.
        The Avian Embryo. Structural and functional development. The Macmillan Compagny, New York1960
        • Romanoff A.L.
        • Romanoff A.J.
        Biochemistry of the avian embryo: A Quantitative Analysis of Prenatal Development. Interscience Publishers, a division of John Wiley & Sons, Inc, New York, London (UK), Sydney (Australia)1967
        • Tian X.
        • Gautron J.
        • Monget P.
        • Pascal G.
        What Makes an Egg Unique? Clues from Evolutionary Scenarios of Egg-Specific Genes.
        Biol. Reprod. 2010; 83: 893-900
        • Clinton M.
        • Haines L.
        • Belloir B.
        • McBride D.
        Sexing chick embryos: a rapid and simple protocol.
        Br. Poult. Sci. 2001; 42: 134-138
        • Shevchenko A.
        • Wilm M.
        • Vorm O.
        • Mann M.
        Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels.
        Anal. Chem. 1996; 68: 850-858
        • Vizcaino J.A.
        • Deutsch E.W.
        • Wang R.
        • Csordas A.
        • Reisinger F.
        • Rios D.
        • Dianes J.A.
        • Sun Z.
        • Farrah T.
        • Bandeira N.
        • Binz P.A.
        • Xenarios I.
        • Eisenacher M.
        • Mayer G.
        • Gatto L.
        • Campos A.
        • Chalkley R.J.
        • Kraus H.J.
        • Albar J.P.
        • Martinez-Bartolome S.
        • Apweiler R.
        • Omenn G.S.
        • Martens L.
        • Jones A.R.
        • Hermjakob H.
        ProteomeXchange provides globally coordinated proteomics data submission and dissemination.
        Nat. Biotechnol. 2014; 32: 223-226
        • Vilella A.J.
        • Severin J.
        • Ureta-Vidal A.
        • Heng L.
        • Durbin R.
        • Birney E.
        EnsemblCompara GeneTrees: Complete, duplication-aware phylogenetic trees in vertebrates.
        Genome Res. 2009; 19: 327-335
        • Dufourny L.
        • Levasseur A.
        • Migaud M.
        • Callebaut I.
        • Pontarotti P.
        • Malpaux B.
        • Monget P.
        GPR50 is the mammalian ortholog of Mel1c: evidence of rapid evolution in mammals. BMC Evol.
        Biol. 2008; 8: 105
        • Guyot N.
        • Labas V.
        • Harichaux G.
        • Chesse M.
        • Poirier J.C.
        • Nys Y.
        • Rehault-Godbert S.
        Proteomic analysis of egg white heparin-binding proteins: towards the identification of natural antibacterial molecules.
        Sci. Rep. 2016; 6: 27974
        • Bhunia A.K.
        • Johnson M.C.
        • Ray B.
        Direct Detection of an antimicrobial peptide of Pediococcus-Acidilactici in sodium dodecyl sulfate polyacrylamide gel electrophoresis J.
        Ind. Microbiol. 1987; 2: 319-322
        • Hamburger V.
        • Hamilton H.L.
        A series of normal stages in the development of the chick embryo.
        J. Morphol. 1951; 88: 49-92
        • Guyot N.
        • Rehault-Godbert S.
        • Slugocki C.
        • Harichaux G.
        • Labas V.
        • Helloin E.
        • Nys Y.
        Characterization of egg white antibacterial properties during the first half of incubation: A comparative study between embryonated and unfertilized eggs.
        Poult. Sci. 2016; 95: 2956-2970
        • Da Silva M.
        • Beauclercq S.
        • Harichaux G.
        • Labas V.
        • Guyot N.
        • Gautron J.
        • Nys Y.
        • Rehault-Godbert S.
        The family secrets of avian egg-specific ovalbumin and its related proteins Y and X.
        Biol. Reprod. 2015; 93: 71
        • Wellman-Labadie O.
        • Lakshminarayanan R.
        • Hincke M.T.
        Antimicrobial properties of avian eggshell-specific C-type lectin-like proteins.
        FEBS Lett. 2008; 582: 699-704
        • Rehault-Godbert S.
        • Labas V.
        • Helloin E.
        • Herve-Grepinet V.
        • Slugocki C.
        • Berges M.
        • Bourin M.-C.
        • Brionne A.
        • Poirier J.-C.
        • Gautron J.
        • Coste F.
        • Nys Y.
        Ovalbumin-related protein X is a heparin-binding Ov-serpin exhibiting antimicrobial activities.
        J. Biol. Chem. 2013; 288: 17285-17295
        • Svensson S.L.
        • Pasupuleti M.
        • Walse B.
        • Malmsten M.
        • Morgelin M.
        • Sjogren C.
        • Olin A.I.
        • Collin M.
        • Schmidtchen A.
        • Palmer R.
        • Egesten A.
        Midkine and pleiotrophin have bactericidal properties: preserved antibacterial activity in a family of heparin-binding growth factors during evolution.
        J. Biol. Chem. 2010; 285: 16105-16115
        • Bourin M.
        • Gautron J.
        • Berges M.
        • Attucci S.
        • Le Blay G.
        • Labas V.
        • Nys Y.
        • Rehault-Godbert S.
        Antimicrobial potential of egg yolk ovoinhibitor, a multidomain kazal-like inhibitor of chicken egg.
        J. Agric. Food Chem. 2011; 59: 12368-12374
        • Dombre C.
        • Guyot N.
        • Moreau T.
        • Monget P.
        • Da Silva M.
        • Gautron J.
        • Rehault-Godbert S.
        Egg serpins: The chicken and/or the egg dilemma.
        Semin. Cell. Dev. Biol. 2017; 62: 120-132
        • Ahlroth M.K.
        • Grapputo A.
        • Laitinen O.H.
        • Kulomaa M.S.
        Sequence features and evolutionary mechanisms in the chicken avidin gene family.
        Biochem. Biophys. Res. Commun. 2001; 285: 734-741
        • Brawand D.
        • Wahli W.
        • Kaessmann H.
        Loss of egg yolk genes in mammals and the origin of lactation and placentation.
        PLos Biol. 2008; 6: e63
        • Metz-Boutigue M.H.
        • Jolles J.
        • Mazurier J.
        • Schoentgen F.
        • Legrand D.
        • Spik G.
        • Montreuil J.
        • Jolles P.
        Human lactotransferrin: amino acid sequence and structural comparisons with other transferrins.
        Eur. J. Biochem. 1984; 145: 659-676
        • Bellamy W.
        • Takase M.
        • Yamauchi K.
        • Wakabayashi H.
        • Kawase K.
        • Tomita M.
        Identification of the bactericidal domain of lactoferrin.
        Biochim. Biophys. Acta. 1992; 1121: 130-136
        • Baron F.
        • Jan S.
        • Gonnet F.
        • Pasco M.
        • Jardin J.
        • Giudici B.
        • Gautier M.
        • Guerin-Dubiard C.
        • Nau F.
        Ovotransferrin plays a major role in the strong bactericidal effect of egg white against the Bacillus cereus group.
        J. Food Prot. 2014; 77: 955-962
        • Mann K.
        The chicken egg white proteome.
        Proteomics. 2007; 7: 3558-3568
        • Mann K.
        • Mann M.
        The chicken egg yolk plasma and granule proteomes.
        Proteomics. 2008; 8: 178-191
        • Palmiter R.D.
        Regulation of protein synthesis in chick oviduct. I. Independent regulation of ovalbumin, conalbumin, ovomucoid, and lysozyme induction.
        J. Biol. Chem. 1972; 247: 6450-6461
        • Nakano T.
        • Graf T.
        Goose-type lysozyme gene of the chicken: sequence, genomic organization and expression reveals major differences to chicken-type lysozyme gene.
        Biochim. Biophys. Acta. 1991; 1090: 273-276
        • Slade B.
        • Milne J.
        Localization and synthesis of alpha-fetoprotein in the chicken.
        Cell Tissue Res. 1977; 180: 411-419
        • Anton M.
        Egg yolk: structures, functionalities and processes.
        J. Sci. Food Agriculture. 2013; 93: 2871-2880
        • Malik G.K.
        • Sinha S.M.
        • Saksena P.N.
        • Kapoor A.K.
        • Mehra P.
        • Bagchi M.
        • Agarwal D.K.
        • Tuteja N.
        Amniotic fluid proteins in relation to fetal maturity. Indian J.
        Pediatr. 1981; 48: 149-152
        • Retzek H.
        • Steyrer E.
        • Sanders E.J.
        • Nimpf J.
        • Schneider W.J.
        Molecular cloning and functional characterization of chicken cathepsin D, a key enzyme for yolk formation.
        DNA Cell Biol. 1992; 11: 661-672
        • Nelson T.C.
        • Groth K.D.
        • Sotherland P.R.
        Maternal investment and nutrient use affect phenotype of American alligator and domestic chicken hatchlings.
        Comp. Biochem. Physiol. A-Mol. Integr. Physiol. 2010; 157: 19-27
        • Maehashi K.
        • Ueda M.
        • Matano M.
        • Takeuchi J.
        • Uchino M.
        • Kashiwagi Y.
        • Watanabe T.
        Biochemical and functional characterization of transiently expressed in neural precursor (TENP) protein in emu egg white.
        J. Agric. Food Chem. 2014; 62: 5156-5162
        • Elo H.A.
        • Korpela J.
        The occurrence and production of avidin: a new conception of the high-affinity biotin-binding protein.
        Comp. Biochem. Physiol. B. 1984; 78: 15-20
        • Matulova M.
        • Rajova J.
        • Vlasatikova L.
        • Volf J.
        • Stepanova H.
        • Havlickova H.
        • Sisak F.
        • Rychlik I.
        Characterization of chicken spleen transcriptome after infection with Salmonella enterica serovar Enteritidis.
        PLoS ONE. 2012; 7: e48101
        • Nakimbugwe D.
        • Massehalck B.
        • Atanassova M.
        • Zewdie-Bosuner A.
        • Michiels C.W.
        Comparison of bactericidal activity of six lysozymes at atmospheric pressure and under high hydrostatic pressure.
        Int. J. Food Microbiol. 2006; 108: 355-363
        • Sellier N.
        • Vidal M.L.
        • Baron F.
        • Michel J.
        • Gautron J.
        • Protais M.
        • Beaumont C.
        • Gautier M.
        • Nys Y.
        Estimations of repeatability and heritability of egg albumen antimicrobial activity and of lysozyme and ovotransferrin concentrations.
        Br. Poult. Sci. 2007; 48: 559-566
        • Herve-Grepinet V.
        • Rehault-Godbert S.
        • Labas V.
        • Magallon T.
        • Derache C.
        • Lavergne M.
        • Gautron J.
        • Lalmanach A.C.
        • Nys Y.
        Purification and Characterization of Avian beta-Defensin 11, an Antimicrobial Peptide of the Hen Egg.
        Antimicrob. Agents Chemother. 2010; 54: 4401-4408
        • Tollin M.
        • Bergsson G.
        • Kai-Larsen Y.
        • Lengqvist J.
        • Sjovall J.
        • Griffiths W.
        • Skuladottir G.V.
        • Haraldsson A.
        • Jornvall H.
        • Gudmundsson G.H.
        • Agerberth B.
        Vernix caseosa as a multi-component defence system based on polypeptides, lipids and their interactions.
        Cell. Mol. Life Sci. 2005; 62: 2390-2399
        • Yoshio H.
        • Tollin M.
        • Gudmundsson G.H.
        • Lagercrantz H.
        • Jornvall H.
        • Marchini G.
        • Agerberth B.
        Antimicrobial polypeptides of human vernix caseosa and amniotic fluid: implications for newborn innate defense.
        Pediatr. Res. 2003; 53: 211-216
        • Cunningham M.
        • Tang J.
        Purification and properties of cathepsin D from porcine spleen.
        J. Biol. Chem. 1976; 251: 4528-4536
        • Castellano A.C.
        • Barteri M.
        • Bianconi A.
        • Bruni F.
        • Della Longa S.
        • Paolinelli C.
        Conformational changes involved in the switch from ovalbumin to S-ovalbumin.
        Z Naturforsch C. 1996; 51: 379-385
        • Yamasaki M.
        • Takahashi N.
        • Hirose M.
        Crystal structure of S-ovalbumin as a non-loop-inserted thermostabilized serpin form.
        J. Biol. Chem. 2003; 278: 35524-35530
        • Spencer K.
        • Muller F.
        • Aitken D.A.
        Biochemical markers of trisomy 21 in amniotic fluid.
        Prenat. Diagn. 1997; 17: 31-37
        • Liu Y.
        • Liu Y.
        • Du C.
        • Zhang R.
        • Feng Z.
        • Zhang J.
        Diagnostic value of amniotic fluid inflammatory biomarkers for subclinical chorioamnionitis.
        Int. J. Gynaecol. Obstet. 2016; 134: 160-164
        • Hsu T.Y.
        • Lin H.
        • Hung H.N.
        • Yang K.D.
        • Ou C.Y.
        • Tsai C.C.
        • Cheng H.H.
        • Chung S.H.
        • Cheng B.H.
        • Wong Y.H.
        • Chou A.K.
        • Hsiao C.C.
        Two-Dimensional differential gel electrophoresis to identify protein biomarkers in amniotic fluid of Edwards Syndrome (Trisomy 18) pregnancies.
        PLoS ONE. 2016; 11: e0145908
        • Wolanski N.J.
        • Renema R.A.
        • Robinson F.E.
        • Carney V.L.
        • Fancher B.I.
        Relationships among egg characteristics, chick measurements, and early growth traits in ten broiler breeder strains.
        Poult. Sci. 2007; 86: 1784-1792
        • Karcher D.M.
        • Jones D.R.
        • Abdo Z.
        • Zhao Y.
        • Shepherd T.A.
        • Xin H.
        Impact of commercial housing systems and nutrient and energy intake on laying hen performance and egg quality parameters.
        Poult. Sci. 2015; 94: 485-501
        • Bakst M.R.
        • Welch G.R.
        • Fetterer R.
        • Miska K.
        Impact of broiler egg storage on the relative expression of selected blastoderm genes associated with apoptosis, oxidative stress, and fatty acid metabolism.
        Poult. Sci. 2016; 95: 1411-1417
        • Loyau T.
        • Hennequet-Antier C.
        • Coustham V.
        • Berri C.
        • Leduc M.
        • Crochet S.
        • Sannier M.
        • Duclos M.J.
        • Mignon-Grasteau S.
        • Tesseraud S.
        • Brionne A.
        • Metayer-Coustard S.
        • Moroldo M.
        • Lecardonnel J.
        • Martin P.
        • Lagarrigue S.
        • Yahav S.
        • Collin A.
        Thermal manipulation of the chicken embryo triggers differential gene expression in response to a later heat challenge.
        BMC Genomics. 2016; 17: 329