Effect of Embryonic Development on the Chicken Egg Yolk Plasma

Article pubs.acs.org/JAFC

Effect of Embryonic Development on the Chicken Egg Yolk Plasma Proteome after 12 Days of Incubation Sophie Réhault-Godbert,*,†,# Karlheinz Mann,‡,# Marie Bourin,†,§ Aurélien Brionne,† and Yves Nys† †

INRA, UR83 Recherches Avicoles, Fonction et Régulation des Protéines de l’Œuf, F-37380 Nouzilly, France Abteilung Proteomics und Signaltransduktion, Max-Planck-Institut für Biochemie, Am Klopferspitz 18, D-82152 Martinsried, Germany

‡

S Supporting Information *

ABSTRACT: 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. Alphafetoprotein and two uncharacterized proteins (F1NHB8 and F1NMM2) were identified for the first time in the egg. After 12 days of incubation, five proteins (vitronectin, α-fetoprotein, similar to thrombin, apolipoprotein B, and apovitellenin-1) 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. KEYWORDS: egg, embryonic development, storage, proteomics, yolk plasma

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INTRODUCTION Egg yolk is known for its high nutritive value for humans, but its biological function in avian reproduction is to provide numerous bioactive components and nutrients for the developing embryo. Most egg yolk components are synthesized in the liver of sexually mature hens. These egg yolk precursors are secreted into the bloodstream and transferred by receptormediated endocytosis1,2 to the yolky ovarian follicles, where they are processed by endogenous proteinase(s).3 Three hundred and sixteen different egg yolk proteins were identified by various complementary proteomic approaches in the laid unfertilized egg (table egg).4−7 The proteins with highest abundance were serum albumin, vitellogenin-derived products, apovitellenins, IgY, and ovalbumin.7 Albumin, the major carrier protein of plasma, and ovalbumin, the major egg white protein, are supposed to provide nutrients for the developing embryo. Phosvitin, a proteolytic product of vitellogenins, is believed to store calcium, iron, and other cations for the embryo.8 Apovitellenin-1 is a low-density lipoprotein lipase inhibitor of egg yolk,9 specifically synthesized by laying hens to protect the highly specific hen very low density lipoproteins from lipase activities in blood plasma, on their way to the follicle.9 Yolk immunoglobulins Y protect the embryo against the attack of pathogens. This passive immunity is of major importance to the embryo, because the immune system of the chick matures only after hatching. The presence and specificity of these molecules in egg yolk depends on the exposure of the laying hens to pathogens and exogenous molecules and may therefore vary in terms of type, concentration and pathogen-specificity between lines,10 individual animals, and between eggs from the same animal. Many other minor proteins have been identified in egg yolk and include proteins also occurring in blood plasma, such as components of coagulation and inflammatory cascades.11 © 2014 American Chemical Society

The biological significance of such minor molecules in egg yolk remains unclear, and the very low abundance of many of them suggests that they appear in yolk as coproducts of the incorporation of specific components and do not have a function in this egg compartment. At present, the biological function of most egg yolk proteins during embryonic development remains to be explored. Chicken embryonic development can be divided into three phases: early embryogenesis, corresponding to the establishment of the embryonic germ and the formation of chorionic sac and allantoic cavity (first week of incubation), embryo completion from day 8 to day 14 that relies on nutrient recovery from albumen and yolk, and the final stage, corresponding to the preparation for emergence. This final stage includes a transfer of remaining egg white into the amniotic sac and a rapid oral absorption of the resulting mixture of nutrients by the embryo.12,13 With regard to the egg white, some studies reported that the overall protein content of egg white is relatively constant during the first 7 days of embryogenesis,14−16 although some changes were observed after incubation up to 17 days.17 Egg white is gradually assimilated by the embryo and almost disappears from day 15 of incubation onward.12 The function of most egg white proteins in embryonic development is still unclear. Indeed, a recent proteomic study on egg white proteins has revealed that only eight proteins out of the 148 identified in egg white showed a significant change in relative abundance during the first week of incubation,15 suggesting that the assimilation of Received: Revised: Accepted: Published: 2531

October 10, 2013 February 25, 2014 March 3, 2014 March 3, 2014 dx.doi.org/10.1021/jf404512x | J. Agric. Food Chem. 2014, 62, 2531−2540

Journal of Agricultural and Food Chemistry

Article

conditions using 12.5% acrylamide-bisacrylamide gels and Coomassie Blue staining. Preparation of Peptides. Reduction, carbamidomethylation, and enzymatic cleavage of yolk proteins were performed using a modified FASP (filter-aided sample preparation) method.25 Fifty microgram aliquots of egg yolk proteins were dissolved in 100 μL of 0.1 M Tris, pH 8, containing 6 M guanidine hydrochloride and 0.01 M dithiothreitol (DTT). This mixture was heated to 56 °C for 60 min, cooled to room temperature, and loaded into a Microcon YM-30 centrifugal filter device (Millipore). DTT was removed by centrifugation at 13 000 rpm in an Eppendorf benchtop centrifuge model 5415D for 10 min and washing with 2 × 1 vol of the same buffer. Carbamidomethylation was done in the device using 0.1 M Tris buffer, pH 8, containing 6 M guanidine hydrochloride and 0.05 mM iodoacetamide and incubation for 45 min in the dark. Carbamidomethylated proteins were washed with 0.05 M ammonium hydrogen carbonate buffer, pH 8, containing 2 M urea, and centrifugation as before. Trypsin (1 μg, sequencing grade, modified; Promega, Madison, WI) was added in 40 μL of the same buffer, and the devices were incubated at 37 °C for 16 h. Peptides were collected by centrifugation, and the filters were washed twice with 40 μL of buffer. The peptide solution was acidified to pH 1−2 with trifluoroacetic acid, and peptides were cleaned and concentrated using five successive C18 Stage Tips.26 The eluate of all five StageTips was combined for mass spectrometric analysis. Mass Spectrometric Analysis and Evaluation. Peptide mixtures were analyzed by LC_MS/MS using an LTQ Orbitrap Velos mass spectrometer (Thermo Fisher Scientific, Bremen, Germany)27 as described before.28 Raw files were processed with MaxQuant (http:// maxquant.org/)29−31 version 1.3.8.1. The initial peptide mass tolerance was set to 20 ppm, and the main search tolerance was set to 6 ppm. The false discovery rate (FDR) for both peptides and proteins was set to 0.01. The maximal posterior error probability (PEP, the probability of each peptide to be a false hit considering identification score and peptide length) was set to 0.01. Carbamidomethylation was set as fixed modification. Variable modifications were oxidation (M), N-acetyl (protein), pyro-Glu/Gln (N-terminus), and deamidation (N,Q). The required minimal peptide length was seven amino acids, and two miss-cleavages were allowed. The Requantify and the Label-Free Quantitation (LFQ) option 32 were enabled, together with the Match between runs option using a time window of 2 min. The minimal ratio count for LFQ was set to 2. The database was a Gallus gallus subset of UniProt release 20012_8, containing 27 027 entries (http://www.uniprot.org/) combined with the reverse sequences and sequences of common contaminants. In general, only protein identifications with more than two sequenceunique peptides distributed in at least two replicates were accepted. Exceptions were proteins sharing peptides with similar proteins or fragments of the same protein. Label-free quantitation required the presence of 12 (of 16 possible) valid LFQ intensity values in at least one group of replicates belonging together, and the presence of at least two peptides in the razor + unique peptides column. The results of label-free quantitation were analyzed using an ANOVA multiplesample test and hierarchical clustering provided by Perseus (http:// maxquant.org/)29−31 version 1.3.8.1. Because not all proteins yielded peptides in all replicates, missing intensity values were imputed using values from a suitable Gaussian distribution for minor proteins. Oneway ANOVA was done with a permutation-based false discovery rate of 0.01 for truncation. Hierarchical clustering was done after Z-scoring to analyze the enrichment among all four samples (Perseus (http:// maxquant.org/)).

egg white proteins is rather nonselective. These included ovalbumin, ovalbumin-related protein Y, clusterin, PG D2 synthase, ovotransferrin, lysozyme, and an uncharacterized protein.15 More recently, ovoinhibitor, extracellular fatty acidbinding protein precursor, and apolipoprotein D precursor were also shown to change in abundance in egg white between day 0 and day 7 of incubation.18 Additionally, proteomic approaches applied to eggshell membranes from day 1 to day 21 of incubation revealed many proteins exhibiting fluctuation during embryonic development, suggesting a possible specific role according to the embryonic stage.19 In contrast, there was no similar comprehensive study on yolk protein modification during incubation, although physicochemical properties of yolk are known to change during embryonic development.20−23 To investigate the dynamics of egg yolk components in relation to their primary role as a nutritional and physiological support for the embryo during development, we analyzed the proteomic profiles of egg yolk plasma soluble proteins isolated from fertilized and unfertilized eggs at the day of lay and after the first 12 days of incubation (at the end of embryo completion). From the proteomics studies published previously,7 it seems that the proteins identified in the watersoluble fraction of egg yolk are representative of most of the proteins identified in egg yolk. Indeed the granular fraction is mostly composed of apovitellin, apolipoprotein, and vitellogenin (cleavage products) in high quantities, proteins which are also found in the water-soluble fraction. We believe that the analysis of the water-soluble fraction is more informative than the analysis of the granular fraction. Unfertilized eggs were used as controls to exclude proteins which undergo changes due to storage, such as temperature and automatic reversal, independently of the presence of an embryo. Therefore, this study examines proteomic changes occurring after incubation of fertilized eggs but also as a result of storage/incubation of unfertilized eggs. The results provide a general view on the utilization of egg yolk components by the embryo during incubation but also indicate how storage at high temperature (accelerated aging) can alter the protein profile of egg yolk of unfertilized eggs.

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MATERIAL AND METHODS

Preparation of Egg Yolk Plasmas. Nonfertilized eggs (ISA Brown, Hendrix Genetics, St Brieuc, France) and eggs fertilized by artificial insemination (INRA, UE1295 Pôle d’Expérimentation Avicole de Tours, F-37380 Nouzilly, France) were collected at the day of lay and 12 days after incubation (45% hygrometry, 37.8 °C, automatic reversal every hour, INRA, UE1295 Pôle d’Expérimentation Avicole de Tours, F-37380 Nouzilly, France). At day 0 (day of lay) and day 12, egg yolks from eight eggs were independently collected, diluted 1:10 (w/v) in demineralized water, acidified to pH 5 with HCl according to Mann et al., 24 and centrifuged 1 h at 10 000g, 4 °C, to pellet the granular fraction of egg yolk. The resulting supernatants were dialyzed against 50 mM Tris-HCl, 50 mM NaCl, pH 7.4 for 24 h at 4 °C. Clarified egg yolks (egg yolk plasmas) were obtained after centrifugation at 4 °C for 1 h at 10 000g. All experiments were conducted according to 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). The protein concentrations of each sample were determined using the Protein Dc Assay (Bio-Rad, Marnes-la-Coquette, France) and bovine serum albumin (Sigma-Aldrich, Saint-Quentin-Fallavier, France) as the standard. All samples were lyophilized and further analyzed by SDS-PAGE under denaturing, but nonreducing,

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RESULTS AND DISCUSSION As a preliminary study, we collected samples of egg yolks at day 0, 4, 8, and 12 of incubation. The first analysis by SDS-PAGE of all our samples, collected and treated under identical conditions, indicated either a high heterogeneity between samples collected at day 4 or 8 and/or no dramatic change as compared with day 0 and day 12, respectively (Figure 1). The 2532

dx.doi.org/10.1021/jf404512x | J. Agric. Food Chem. 2014, 62, 2531−2540


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in the following sections. Other proteins, ADP-ribosyl cyclase CD157, gamma-glutamyl hydrolase, apolipoprotein A-IV, 14− 3−3 zeta protein and chondrogenesis-associated lipocalin, ovosecretoglobulin, and mucin 5B, were previously identified in other egg compartments4,5,7,11,16,28,34−37 but were identified for the first time as yolk components in the present study. Only three minor proteins were apparently identified in the chicken egg for the first time. These were α-fetoprotein, a major plasma protein produced by the yolk sac and the liver during fetal life,38 an uncharacterized peptidase belonging to the C1 family (F1NHB8), and an uncharacterized protein with predicted Chitinase activity (F1NMM2), which is highly expressed in the stomach and the liver of chickens.39 The presence of this latter protein in the yolk is consistent with the hepatic origin of yolk constituents. Figure 2 shows a typical annotated MS/MS spectrum for each of these new egg proteins. For label-free quantitation, the protein distribution was analyzed with ANOVA to detect significant differences among the four sets of biological replicates, FE_D0 (fertilized, day 0), FE_D12 (fertilized, day 12), UFE_D0 (unfertilized, day 0), UFE_D12 (unfertilized, day 12). The thresholds defined in section 2.3 left 81 protein groups for quantitation. Seventythree of these protein groups, likely representing a maximum of 69 individual proteins, showed significant differences among the sets of replicates (Supplementary Table 4). Hierarchical clustering (Figure 3) of the data was performed to group proteins with similar change in relative abundance according to the nature of the sample (unfertilized or fertilized) and the length of incubation (D0 and D12). Four clusters, C1 to C4, were defined (Figure.3) and will be described in the following sections. Cluster 1: Proteins with Increased Relative Abundance in Fertilized Eggs after 12 Days of Incubation. Hierarchical clustering revealed five proteins that were significantly enriched in the proteome of yolks of fertilized eggs after 12 days of incubation as compared to other conditions (Cluster 1, Table 1, Figure 3). These were vitronectin, α-fetoprotein, F2/thrombin, apolipoprotein B (distributed over several entries), and apovitellenin-1. One of these proteins, α-fetoprotein (GeneID: 422652, Table 1, bold), was a previously unidentified component of the unfertilized/ table egg. α-Fetoprotein may play a role in the development of the avian immune system.40 It is present in a wide range of chicken embryonic tissues, especially in yolk sac, liver, and connective tissues.38 The significant increase of α-fetoprotein in yolk plasma of 12 day-old eggs is in accordance to previous evidence of a gradual increase in the yolk sac that is then followed by a decline in its synthesis starting at day 14.41 Apolipoprotein B and apovitellenin-1 are major proteins of the granular fraction of the yolk and the cell adhesion and cell spreading protein vitronectin is a relatively minor protein of this fraction.7 Vitronectin is a glycosaminoglycan-binding protein found in serum and tissues, which acts as a cell adhesion and spreading factor. This protein was reported to be a major cell adhesion factor in early chicken embryogenesis.42 The higher relative abundance of these proteins in yolk plasma after incubation may result from solubilization of these proteins from the granules along with the decrease of yolk pH observed during incubation.43 Apolipoprotein B fragments and apovitellenin-1 are major components of very low-density lipoprotein (VLDL) particles, and their enhanced appearance in the soluble fraction may reflect degradation of LDL in the course of lipid recruitment by the growing embryo.

Figure 1. SDS-PAGE analysis of egg yolks from unfertilized and fertilized eggs at 0, 4, 8, and 12 days of incubation. Samples (10 μg) were prepared as described in Material and Methods.

most dramatic changes in protein band pattern were observed between day 0 and day 12 of incubation (Figure 1). From this preliminary study, we decided to compare the proteomes of egg yolk samples from fertilized and unfertilized eggs (as a negative control reflecting the absence of an embryo) at the day of lay and after 12 days of incubation. Of each of the four conditions, eight eggs were used for yolk plasma preparation, and each sample was independently analyzed twice. The resulting 64 raw files were combined for protein identification with MaxQuant. This resulted in the identification of 127 proteins that were distributed into 143 groups by MaxQuant (Supplementary Table S1). The reason for the discrepancy between protein groups and proteins was the occurrence in the database of fragments of the same protein in different entries and entries containing almost identical sequences. These latter may represent variants or isoforms containing possible microheterogeneities or may be due to sequencing errors. For these same reasons, the number of identified proteins may not be absolutely correct. Additional information about the protein groups, such as number of proteins in a group, additional accession numbers, peptide counts in different replicates, sequence coverage, and LTQ intensities, is provided in Supplementary Table 2. Peptide sequences and relevant data about peptides, such as distribution among replicates, scores, and posterior error probability (PEP), are contained in Supplementary Table 3. Both files also contain protein identifications not accepted because they did not meet the quality criteria, for instance, identifications with only one unique peptide. Contaminant entries and reverse hits were removed, except for contaminant peptides shared with chicken proteins. Twenty-one of the protein groups were not identified in yolk in previous proteomic studies (Supplementary Table 1). Ten of these were immunoglobulins, a well-known fraction of the yolk proteome that varies in composition depending on hen environment. Chicken immunoglobulins (IgY) are transferred from the blood of the hen to the yolk by oocyte membrane receptors.10 During incubation, IgY molecules are exported from the embryomic yolk sac into the blood of the embryo after binding to the chicken yolk sac IgY receptor by a pHdependent mechanism.33 This passive immunity is of major importance for the developing embryo because its immune system matures only after hatching. Presence and concentration of IgY molecules depend on the exposure of the mother to pathogenic and nonpathogenic microbes. Because the laying hens used in this study were not bred under controlled or sterile conditions, IgY profiles were not considered as relevant 2533



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Figure 2. Typical MS/MS spectra of peptides instrumental in the identification of three new minor egg proteins. (A) Spectrum of an α-fetoprotein peptide. This doubly charged peptide was identified with a PEP of 0.0003 and a mass error of −0.75 ppm. (B) Spectrum of a triply charged peptide from F1NHB8_CHICK with a PEP of 0.005 and a mass error of −0.2 ppm. (C) Rather complex spectrum of a long doubly charged peptide of F1NMM2_CHICK with a PEP of 4.9 × 10−31 and a mass error of −2 ppm. 2534



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Figure 3. Hierarchical clustering of egg yolk plasma proteins. Clusters C1 to C4 are indicated.

Table 1. Proteins with Increased Abundance in 12 Day-Old Yolks from Fertilized Eggs (Cluster 1; Z-Scoring, Figure 3) protein IDs

gene ID

protein description

E1C7A7;F1NER8;O12945 E1BV96;E1C1G2;P84407 F1NXV6;Q91001 Q90WR3 P11682 F1NV02;F1NRG7 Q197 × 2;Q7LZ77 P02659

395935 422652 395306 419076 396535

vitronectin α-fetoprotein uncharacterized/thrombin hemopexin (fragment) apolipoprotein B (fragment) apolipoprotein B (fragment) apolipoprotein B apovitellenin-1

396476

localization ES, VM, EY ES, EY ES, EW, VM, EY ES, EW, VM, EY

ES, EW, VM, EY

ES, eggshell; EW, egg white; VM, vitelline membrane; EY, egg yolk. Protein that was identified in egg yolk for the first time is shown in bold.

5B, were identified in egg yolk for the first time (Table 2, bold). Several of these proteins were shown to decrease concomitantly in yolk of fertilized eggs upon incubation and may therefore be assimilated by the embryo during its development. These include the two protease inhibitors cystatin and ovoinhibitor, which are likely to regulate the overall proteolytic activity within the yolk compartment, but also ovotransferrin, mucin 5B, and ovoglycoprotein (Figure 3). This group also contains extracellular fatty acid-binding protein (Ex-FABP) and

Cluster 2: Proteins with Increased Relative Abundance in Unfertilized Eggs after 12 Days of Incubation. Seventeen proteins of cluster 2 (C2) showed a significant increase in relative abundance in yolks of unfertilized eggs as compared to fertilized eggs after 12 days of incubation (Table 2, Figure 3). Most of these proteins were previously described as major proteins of the egg white and the vitelline membrane,16,36 but three of them, uncharacterized/ovosecretoglobulin, prostaglandin H2-D isomerase precursor, and mucin 2535



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Table 2. Proteins with Increased Abundance in 12 Day-Old Yolks from Unfertilized Eggs (Cluster 2; Z-Scoring, Figure 3) protein ID E1BSY0 P01012 P01005;B6 V1G0;P52267;Q6LDY9 I0J179 I0J173 F1N8Q8;F1NGP2;Q9YGP0 P01013 ; E1BTF4 F1NU63;F1NJA6;F1NJA7;E1C546; F1NJA5;P20740;F1NJB1;Q9PSS0 B8YK75;B8YK77;B8YK79;P00698; B8YJN9;B8YJP1;B8YJT7;B8YK73;Q6LEL2 P01038 F1NFY4;F1NMN2;F1NUT1;Q6XLT0 P02789 Q8QFM7\;E1BTX1 F1NZY2 E1BTW6;E1C0K3;E1C0K0;E1C0K1; P21760;F6JXX0;F6JXW5;F6JXX6 A7UEB0;Q8JIG5;F1P2P7;E1BZ67

gene ID

protein description

localization

ENSGALG00000024467 396058 416236 420897 395882 395722 420898 396151

uncharacterized/ovosecretoglobin ovalbumin ovomucoid ovalbumin-related Y ovoglobulin G2/TENP clusterin ovalbumin-related protein X ovostatin

EW ES, EW, ES, EW, ES, EW, ES, EW, ES, EW, ES, EW, ES, EW,

396218

lysozyme C

ES, EW, VM, EY

396497 416235 396241 374110 395381 396393

cystatin ovoinhibitor ovotransferrin prostaglandin-h2 D-isomerase precursor mucin 5B extracellular fatty acid-binding protein

ES, EW, VM, ES, EW, VM, ES, EW, VM, ES, EW, VM ES, EW, VM ES, EW, VM,

395220

alpha1-acidic glycoprotein/ovoglycoprotein

ES, EW, VM, EY

VM, VM, VM, VM, VM, VM, VM,

EY EY EY EY EY EY EY

EY EY EY

EY

ES, eggshell; EW, egg white; VM, vitelline membrane; EY, egg yolk. Proteins that were identified in egg yolk for the first time are shown in bold.

Table 3. Proteins with Decreased Abundance in 12day-Old Yolks from Unfertilized Eggs (Cluster 3, Z-Scoring, Figure 3) protein ID

gene ID

protein description

localization

H9KZU1 Q90633;A6N9E0;Q2MV09;H9L2W3 F4YMA7;F1NHT7;O42486;F1NJV8;E1C1 V3;E1C2U5;Q6PVZ8 F1NFL6;E1BYN6;F1NV01 E1BWI0 F1NWX6;F1NTX4

ENSGALG00000011667 396370 429710 424533 420869 421580

uncharacterized/similar to natterin complement C3 plakoglobin vitellogenin-2 uncharacterized/desmoplakin uncharacterized/plasminogen

EY ES, EW, VM, EY EW, EY ES, EW, VM, EY ES, EW, VM, EY EY

ES, eggshell; EW, egg white; VM, vitelline membrane; EY, egg yolk.

Cluster 3 contained four proteins exhibiting significant change that were present in both fertilized and unfertilized yolks but decrease in relative abundance specifically in egg yolks of unfertilized eggs during incubation (Table 3, Figure 3). These were a protein similar to the poison toadfish protease natterin, complement factor C3, the junction plaque component plakoglobin, and an uncharacterized protein similar to the desmosomal protein desmoplakin. All four proteins were previously identified as minor yolk components, and their role in egg yolk remains unexplored. Their distribution in yolk replicate groups indicated a loss in unfertilized eggs due to aging. Cluster 4: Proteins with Decreased Relative Abundance after 12 Days of Incubation of Fertilized and Unfertilized Eggs. Cluster 4 (C4) comprised 43 proteins with a significant decrease in relative abundance after 12 days of incubation in both fertilized and unfertilized eggs (Figure 3, Table 4), including several immunoglobulin chains identified in the egg yolk for the first time. We suspect that the mechanism by which these proteins are decreasing in fertilized versus unfertilized eggs might be different because the presence of an embryo is likely to affect yolk proteins. If we focus on fertilized eggs, this cluster contained several proteins known to play a role in embryonic development, such as the vitamin-A-binding retinol-binding protein 4.44,45 Retinolbinding protein 4 has been associated with many biological processes related to embryonic development, 49 including morphogenesis (GO:0048562) and development of heart (GO:0001555; GO:0060347, GO:0007507; GO:0048738,

prostaglandin H2-D isomerase precursor, which belong to the lipocalin protein family and are encoded in genes located within the same genomic locus. Both proteins have been shown to be expressed ubiquitously by embryo tissues with especially high levels of mRNA transcripts in liver and brain.44,45 Ex-FABP is also associated with heart (GO:0007507) and muscle development (GO:0010831, GO:0045663, GO:0048747), as suggested by Gene Ontology or GO terms (www.geneontology.org). The decreased relative abundance of both proteins in yolks of fertilized eggs after 12 days of incubation may be related to organ development in the embryo. The observed increase in relative abundance of most of these proteins as compared to proteomes of nonincubated eggs may be due to storage at relatively high temperature (37.8 °C). Deterioration of the perivitelline membrane46,47 and thinning of egg white upon aging 48 may facilitate the migration of egg white proteins into yolk and thus contribute to the observed increase in relative abundance. To date, the mechanism by which these proteins accumulate in the egg yolk of unfertilized eggs during incubation remains unexplained. No significant relationship between the physicochemical properties of these proteins (molecular mass/pI) and their increased relative abundance in the egg yolk could be found. Approximately 70% of these proteins exhibit an isoelectric point of 5−7 and a molecular weight of 10−50 kDa. However, lysozyme has a pI of about 9, and ovostatin has a molecular weight above 150 kDa (data not shown). Cluster 3: Proteins with Decreased Relative Abundance in Unfertilized Eggs after 12 Days of Incubation. 2536



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Table 4. Proteins with Decreased Abundance in Egg Yolks after 12 Days of Incubation (Cluster 4, Z-Scoring, Figure 3) protein IDs F1N904 F1NK40 ; H1AC38 H9KZK6;Q98TD1 E1BZE1 E1C7D5 P81475 F2Z4L6;P19121 F1NXM7 E1C7P4 F1NSD0;P20763;P04210 E1BX43 F1NUL9;Q02020;F1P5F9;B4ZCM0;CON__P02676 P41263 F1NHI4 F1NC22 F1NIN2;Q5ZIJ5 E1BUA6 H9L1N5\;H9KZF6 H9KZC7\;H9KZC6 F1P4 V1;P14448 Q5ZHM4;F1P4I7;E1BUA7 E1BYN5;E1BZD1 F1NYG4;E1BSE0 F1NDH2 H9L2S7 O93510;F1NKF3 F1NBX7 F1NF64 F1NSM9;O93568;E1BV78 H9L1N7 F1NSD3 F1P580 E1BS56 F1NI04 P27731;F1NMT4 E1BY96;F1P350 F1NEQ4 P08250;F1N8F3;H9KZR3 H9KZD6 H9KZC5;H9KZT0 F1P587 O73840;F1NJ16 F1NQF3;F1N9N4;Q5ZJS7 P02752;Q9PWK0;F1NX08;Q7SX70 A2N883\;A2N881\;A2N884\;A2N885

gene ID

protein description

396197 420302 429057 416928 424957 373926 396166 422810 ENSGALG00000005937 428163 421702 ENSGALG00000022049 ENSGALG00000005880 396307 421702 424534 417431 421543 ENSGALG00000022419 395774 ENSGALG00000005946 428773 395837 ENSGALG00000022053 769305 423629 423433 417829 396277 424547 427942 396536 ENSGALG00000022501 ENSGALG00000022031 418892 395877 423353 396449 776783

IGv fragment uncharacterized/similar to alpha2-macroglobulin uncharacterized/PIT54 alpha2-HS-glycoprotein uncharacterized/similar to transthyretin complement factor B-like protease serum albumin uncharacterized/similar to prostate stem cell antigen uncharacterized/complement factor H Ig lambda chain C region uncharacterized/kininogen-1 fibrinogen beta-chain;fibrinopeptide B;fibrinogen beta chain retinol-binding protein 4 superoxide dismutase [Cu−Zn] IGv fragment uncharacterized/cathepsin A uncharacterized/vanin-1 IGv fragment IGv fragment fibrinogen alpha-chain;fibrinopeptide A;fibrinogen alpha chain uncharacterized/similar to vanin-2 vitellogenin-3 uncharacterized/apolipoprotein H sngiotensinogen IGv fragment Gelsolin IGv fragment uncharacterized/complement factor I fibrinogen gamma-chain IGv fragment IGv fragment uncharacterized/LOC423629 uncharacterized/alpha-1-antiproteinase 2 uncharacterized/N-acetylglucosamine-6-sulfatase transthyretin uncharacterized/vitellogenin-1 uncharacterized/similar to alpha2-macroglobulin apolipoprotein A-I IGv fragment IGv fragment uncharacterized/similar to complement C4−1 heparin cofactor II uncharacterized/epididymal secretory protein E1 precursor riboflavin-binding protein IGv fragment

769327 427942 395364 424956 421091

localization EY ES, EY ES, EY ES, EY EY ES, EW, VM, EY ES, EY EY EW, VM, EY EY ES, EY ES, EY EY ES, EW, VM, EY ES, EY EY

EY EY ES, EY ES, EY ES, EY EY ES, EY EW, EY EY EY

ES, EY ES, ES, ES, EY ES, ES, EY ES, ES, ES, ES,

EY EY EY EW, VM, EY EW, VM, EY VM, EY EW, EW, EW, EW,

EY EY EY VM, EY

ES, eggshell; EW, egg white; VM, vitelline membrane; EY, egg yolk. Proteins that were identified in egg yolk for the first time are shown in bold.

GO:006004 50), lung (GO:0030324), eye (GO:0001654, GO:0060041, GO:0060059), the skeletal system (GO:0048706), the urinary system (GO:0060157), and gonads (GO: 0008584; GO:0048807; GO:0060065; GO:0060068; GO:0060068). Its decrease in yolk of fertilized eggs during incubation suggests that it might be selectively used to for embryonic development. Similarly, we observed a marked reduction in transthyretin abundance in yolk of fertilized egg as compared to other conditions. This protein is known to interact with retinol-binding protein. Our data are consistent with previous observations suggesting a role for transthyretin (previously called prealbumin) in the transport and distribution of thyroid hormone from yolk to embryo prior to establishment of the circulatory system.50 Yolk riboflavin-binding protein is synthesized by the liver in response to estrogen,51 secreted into

the blood and further transferred to the growing follicles.52 During incubation, this protein is removed from yolk and egg white and accumulates in embryonic tissues in a complex with riboflavin up to the fourteenth day of incubation.53 This accumulation in tissues coincides with the disappearance of riboflavin-binding protein from the white and the yolk compartments, 53 which is consistent with the decrease in relative abundance we observed in egg yolk after 12 days of incubation. Superoxide dismutases [Cu-Zn] are known to destroy radicals that are normally produced within the cells and are toxic to biological systems. This antioxidant has been shown to regulate angiogenesis using the model of the chicken embryo chorioallantoic membrane.54 Proteins which are decreasing in unfertilized eggs in egg yolk plasma during incubation are mostly “egg-yolk-specific” 2537



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inappropriate storage conditions. This work has shown that many proteins initially identified in the egg white and the vitelline membrane increase in relative abundance in the egg yolk after 12 days (Cluster 2). In contrast, decreases in relative abundance could be due either to endogenous degradation by proteases or diffusion of yolk plasma proteins to egg white or the yolk granular fraction upon aging. It is known that the perivitelline membrane not only deteriorates during embryonic development in fertilized eggs but also in stored unfertilized eggs. At the same time, the egg white of unfertilized eggs show the well-known thinning that may be the consequence of reorganization of egg white protein interactions and which could enhance diffusion of proteins to the other compartments. The relatively high temperature used in this study may enhance such processes. Furthermore, the loss of certain proteins may apparently increase the relative abundance of others.

proteins (Table 4). The decrease of these proteins in yolk plasma after 12 days of incubation can be explained by at least three hypotheses: (1) endogenous degradation, (2) interaction with lipoproteins and their further relocalization to the granular fraction of the yolk, and (3) diffusion toward the egg white or other compartments. With regard to the first hypothesis, we know that several proteases are present in egg yolk, including aspartic proteases such as “similar to nothepsin”,5,7,55 many proteases involved in inflammatory cascades including thrombin and coagulation factors,11 and matrix metalloproteinase 2.56 These various proteases could regulate the activity of some egg yolk proteins and their subsequent degradation. Alternatively, we cannot exclude that the proteins identified in the plasma fraction at the day of lay are not recovered in this plasma fraction after incubation. Indeed, the texture and the chemical modification during incubation might affect the solubility of these proteins, which could then associate with the granular fraction of the egg yolk rather than with the plasma. Another explanation is related to the possible exchange of material between egg yolk and egg white during incubation at 37.8 °C. No significant increase in egg yolk proteins has been reported in the egg white of table eggs (unfertilized eggs) stored at ambient temperature up to 40 days.48 However, the conditions of incubation used in our assay, a higher temperature together with the control of relative humidity and automatic egg turning, might slightly modify the dynamics of protein exchange and the physicochemical nature of egg proteins. To our knowledge, this study is the most detailed work available at present on the change of egg yolk protein content occurring during early embryogenesis. In the present study, we have analyzed the effect of fertilization on the yolk plasma proteome after 12 days of incubation and tried to discern changes due to embryonic development from changes due to the aging processes by comparing yolks of fertilized to yolks of unfertilized eggs incubated under the same conditions. The latter results may also be applicable to the storage of table eggs although the conditions used (37.8 °C, constant relative humidity, automatic turning) were not the same as commonly used in supermarkets or homes. We suggest that these conditions may reflect some kind of accelerated aging. It turned out that in many instances, the influence of embryo development and aging overlapped to some degree with most of the identified proteins (Cluster 4). Although the decrease in relative abundance of many proteins in yolks of fertilized eggs may preferentially be attributed to their use by the developing embryo, especially if the decrease is more pronounced in fertilized eggs than nonfertilized eggs, decreases in relative abundance more pronounced in unfertilized eggs (Cluster 3) or increases under certain conditions (Clusters 1 and 2) are more difficult to explain. This analysis performed on the watersoluble fraction of egg yolk (plasma) revealed a likely interaction between yolk plasma and granules, as some proteins might be gradually solubilized from granules during incubation. We also found several proteins, including antiproteases and chelators, that were significantly less abundant after 12 days of incubation as compared to other conditions, providing some clues to study more precisely the kinetics of absorption of some specific egg proteins by the embryo and the regulation of absorption in relation to embryonic stages. Additionally, the study on unfertilized eggs may help to understand possible connections between egg compartments during storage of table eggs and how egg quality can be affected when using

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ASSOCIATED CONTENT

S Supporting Information *

Supplementary Table 1: identified and accepted proteins (protein groups). Supplementary Table 2: MaxQuant protein groups. Supplementary Table 3: MaxQuant peptides. Supplementary Table 4: quantified proteins (ANOVA). This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Present Address §

Institut Technique de l’aviculture, UR83 Recherches Avicoles, F-37380 Nouzilly, France Author Contributions #

These authors contributed equally.

Funding

This research was supported by the French National Research Agency (OVO-mining, ANR-09-BLAN-0136), Region Centre and INRA, Physiologie Animale et Système d’Elevage division, for the financial support of M. Bourin’s PhD. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors acknowledge the experimental unit PEAT (INRA, UE1295 Pôle d’Expérimentation Avicole de Tours, F-37380 Nouzilly, France) and more particularly, Joël Delaveau, for incubating unfertilized and fertilized eggs.

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REFERENCES

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Effect of Embryonic Development on the Chicken Egg Yolk Plasma

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