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Dimorphism between male and female embryos has been demonstrated in many animal species, including chicken species. Likewise, extraembryonic membranes such as the chorioallantoic membrane (CAM) are likely to exhibit a sex-specific profile. Analysis of the previously published RNA-seq data of the chicken CAM sampled at two incubation times, revealed 783 differentially expressed genes between the CAM of male and female embryos. The expression of some of these genes is sex-dependant only at one or other stage of development, while 415 genes are sex-dependant at both developmental stages. These genes include well-known sex-determining and sex-differentiation genes (DMRT1, HEGM, etc.), and are mainly located on sex chromosomes. This study provides evidence that gene expression of extra-embryonic membranes is differentially regulated between male and female embryos. As such, a better characterisation of associated mechanisms should facilitate the identification of new sex-specific biomarkers., Competing Interests: Declaration of Competing Interest The authors declare that they have no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Nutrient availability in eggs can affect early metabolic orientation in birds. In chickens divergently selected on the Pectoralis major ultimate pH, a proxy for muscle glycogen stores, characterization of the yolk and amniotic fluid revealed a different nutritional environment. The present study aimed to assess indicators of embryo metabolism in pHu lines (pHu+ and pHu-) using allantoic fluids (compartment storing nitrogenous waste products and metabolites), collected at days 10, 14 and 17 of embryogenesis and characterized by 1H-NMR spectroscopy. Analysis of metabolic profiles revealed a significant stage effect, with an enrichment in metabolites at the end of incubation, and an increase in interindividual variability during development. OPLS-DA analysis discriminated the two lines. The allantoic fluid of pHu- was richer in carbohydrates, intermediates of purine metabolism and derivatives of tryptophan-histidine metabolism, while formate, branched-chain amino acids, Krebs cycle intermediates and metabolites from different catabolic pathways were more abundant in pHu+. In conclusion, the characterization of the main nutrient sources for embryos and now allantoic fluids provided an overview of the in ovo nutritional environment of pHu lines. Moreover, this study revealed the establishment, as early as day 10 of embryo development, of specific metabolic signatures in the allantoic fluid of pHu+ and pHu- lines., (© 2023. The Author(s).)

The chicken chorioallantoic membrane (CAM) is an extraembryonic membrane that is vital for the embryo. It undergoes profound cell differentiation between 11 and 15 days of embryonic incubation (EID), which corresponds to the acquisition of its physiological functions. To gain insight into the functional genes that accompany these biological changes, RNA sequencing of the CAM at EID11 and EID15 was performed. Results showed that CAM maturation coincides with the overexpression of 4225 genes, including many genes encoding proteins involved in mineral metabolism, innate immunity, homeostasis, angiogenesis, reproduction, and regulation of hypoxia. Of these genes, some exhibit variability in expression depending on the chicken breed (broiler versus layer breeds). Besides the interest of these results for the poultry sector, the identification of new functional gene candidates opens additional research avenues in the field of developmental biology., Competing Interests: Declaration of Competing Interest The authors declare that they have no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

The avian egg perivitelline layer (PL) is a proteinaceous structure that encloses the egg yolk. It consists of the inner and the outer perivitelline layers (IPL and OPL, respectively) that are assumed to play distinct roles in bird reproduction. To gain insight into their respective function, we analyzed the proteome of IPL and OPL in chicken unfertilized eggs after mechanical separation, using a GeLC-MS/MS strategy. Of the 412 proteins identified, 173 proteins were uniquely recovered in IPL and 98 proteins in OPL, while 141 proteins were identified in both sublayers. Genes coding the most abundant proteins were shown to be expressed either in the liver/ovary (IPL formation) or in the oviduct (OPL formation), but rarely in both. The presence of oviduct-specific proteins (including LYZ, VMO1, AvBD11, PTN, OVAL and LOC10175704) in IPL strongly suggests that they participate in the physical association of IPL to OPL, whose tight attachment was further evidenced by analyses of IPL/OPL interfaces (by scanning electron microscopy). Functional annotation of identified proteins revealed functions associated with fertilization and early development for IPL, while OPL would rather participate in egg defense and embryogenesis. Collectively, our data highlight the complementary functions of IPL and OPL that are major determinants of bird reproductive success. SIGNIFICANCE: The present study unveils for the first time the individual proteomes of the two sublayers composing the chicken egg perivitelline layer (PL), which allowed to assign their respective putative biological roles in avian reproduction. The combination of proteomics with gene expression and ultrastructural analyses provides insightful data on the structure and biochemistry of the avian PL. The functional annotation of PL proteins highlights the multifaceted biological functions of this structure in reproduction including fertilization, embryonic development, and antimicrobial protection. This work will stimulate further research to validate predicted functions and to compare the physiology and the functional specificities of PL in egg-laying species., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

The pHu+ and pHu- lines, which were selected based on the ultimate pH (pHu) of the breast muscle, represent a unique model to study the genetic and physiological controls of muscle energy store in relation with meat quality in chicken. Indeed, pHu+ and pHu- chicks show differences in protein and energy metabolism soon after hatching, associated with a different ability to use energy sources in the muscle. The present study aimed to assess the extent to which the nutritional environment of the embryo might contribute to the metabolic differences observed between the two lines at hatching. Just before incubation (E0), the egg yolk of pHu+ exhibited a higher lipid percentage compared to the pHu- line (32.9% vs. 27.7%). Although 1 H-NMR spectroscopy showed clear changes in egg yolk composition between E0 and E10, there was no line effect. In contrast, 1 H-NMR analysis performed on amniotic fluid at embryonic day 10 (E10) clearly discriminated the two lines. The amniotic fluid of pHu+ was richer in leucine, isoleucine, 2-oxoisocaproate, citrate and glucose, while choline and inosine were more abundant in the pHu- line. Our results highlight quantitative and qualitative differences in metabolites and nutrients potentially available to developing embryos, which could contribute to metabolic and developmental differences observed after hatching between the pHu+ and pHu- lines., (© 2022. The Author(s).)

During chicken embryonic development, skeleton calcification mainly relies on the eggshell, whose minerals are progressively solubilized and transported to the embryo via the chorioallantoic membrane (CAM). However, the molecular components involved in this process remain undefined. We assessed eggshell demineralization and calcification of the embryo skeleton after 12 and 16 d of incubation, and analyzed the expression of several candidate genes in the CAM: carbonic anhydrases that are likely involved in secretion of protons for eggshell dissolution (CA2, CA4, CA9), ions transporters and regulators (CALB1, SLC4A1, ATP6V1B2, SGK1, SCGN, PKD2) and vitamin-D binding protein (GC). Our results confirmed that eggshell weight, thickness, and strength decreased during incubation, with a concomitant increase in calcification of embryonic skeletal system. In the CAM, the expression of CA2 increased during incubation while CA4 and CA9 were expressed at similar levels at both stages. SCL4A1 and SCGN were expressed, but not differentially, between the two stages, while the expression of ATP6V1B2 and PKD2 genes decreased. The expression of SGK1 and TRPV6 increased over time, although the expression of the latter gene was barely detectable. In parallel, we analyzed the expression of these candidate genes in the yolk sac (YS), which mediates the transfer of yolk minerals to the embryo during the first half of incubation. In YS, CA2 expression increases during incubation, similar to the CAM, while the expression of the other candidate genes decreases. Moreover, CALB1 and GC genes were found to be expressed during incubation in the YS, in contrast to the CAM where no expression of either was detected. This study demonstrates that the regulation of genes involved in the mobilization of egg minerals during embryonic development is different between the YS and CAM extraembryonic structures. Identification of the full suite of molecular components involved in the transfer of eggshell calcium to the embryo via the CAM should help to better understand the role of this structure in bone mineralization., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: At sexual maturity, the liver of laying hens undergoes many metabolic changes to support vitellogenesis. In published transcriptomic approaches, hundreds of genes were reported to be overexpressed in laying hens and functional gene annotation using gene ontology tools have essentially revealed an enrichment in lipid and protein metabolisms. We reanalyzed some data from a previously published article comparing 38-week old versus 10-week old hens to give a more integrative view of the functions stimulated in the liver at sexual maturity and to move beyond current physiological knowledge. Functions were defined based on information available in Uniprot database and published literature., Results: Of the 516 genes previously shown to be overexpressed in the liver of laying hens, 475 were intracellular (1.23-50.72 fold changes), while only 36 were predicted to be secreted (1.35-66.93 fold changes) and 5 had no related information on their cellular location. Besides lipogenesis and protein metabolism, we demonstrated that the liver of laying hens overexpresses several clock genes (which supports the circadian control of liver metabolic functions) and was likely to be involved in a liver/brain/liver circuit (neurotransmitter transport), in thyroid and steroid hormones metabolisms. Many genes were associated with anatomical structure development, organ homeostasis but also regulation of blood pressure. As expected, several secreted proteins are incorporated in yolky follicles but we also evidenced that some proteins are likely participating in fertilization (ZP1, MFGE8, LINC00954, OVOCH1) and in thyroid hormone maturation (CPQ). We also proposed that secreted proteins (PHOSPHO1, FGF23, BMP7 but also vitamin-binding proteins) may contribute to the development of peripheral organs including the formation of medullar bones to provide labile calcium for eggshell formation. Thirteen genes are uniquely found in chicken/bird but not in human species, which strengthens that some of these genes may be specifically related to avian reproduction., Conclusions: This study gives additional hypotheses on some molecular actors and mechanisms that are involved in basic physiological function of the liver at sexual maturity of hen. It also revealed some additional functions that accompany reproductive capacities of laying hens, and that are usually underestimated when using classical gene ontology approaches.

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., (© 2019 by The American Society for Biochemistry and Molecular Biology, Inc.)

The integrated innate immune features of the calcareous egg and its contents are a critical underpinning of the remarkable evolutionary success of the Aves clade. Beginning at the time of laying, the initial protective structures of the egg, i.e., the biomineralized eggshell, egg-white antimicrobial peptides, and vitelline membrane, are rapidly and dramatically altered during embryonic development. The embryo-generated extra-embryonic tissues (chorioallantoic/amniotic membranes, yolk sac, and associated chambers) are all critical to counteract degradation of primary egg defenses during development. With a focus on the chick embryo (Gallus gallus domesticus), this review describes the progressive transformation of egg innate immunity by embryo-generated structures and mechanisms over the 21-day course of egg incubation, and also discusses the critical interplay between autonomous development and maternal anticipation., (© 2018 The Author(s) Published by S. Karger AG, Basel.)

Twenty-seven serpins belonging to clade A, B, C, D, E, F, G, H and I serpins are currently referenced in chicken genome databases. Phylogenetic analysis of chicken serpins revealed that ovalbumin (Serpinb14) and its paralogs ovalbumin-related protein Y (Serpinb14b) and ovalbumin-related protein X (Serpinb14c) are found in bird species. These clade B serpins are specifically expressed in reproductive tissues and exported in the egg where they constitute major protein components. These data suggest that these three paralogs have probably appeared in birds to face new environments and ensure the extra-uterine development of an embryo in a shell egg. Twelve other serpins have been identified in the newly produced egg, some of them having a specific distribution in the respective egg structures (eggshell, egg white, vitelline membrane and egg yolk). The physiological role of these egg serpins remain largely unexplored, but there is increasing evidence in literature or by homologies with their mammalian counterparts, that some of them participate in cell proliferation, tissue remodeling and/or angiogenesis associated with folliculogenesis and development of extraembryonic structures, eggshell biomineralization, egg defense and nutrition of the embryo. A better knowledge of the phylogenetic evolution of these 15 serpins in other oviparous species, on their egg distribution, on their regulation during embryonic development (activation/degradation/transfer) and on their functional specificity, is needed to better appreciate their role and their bird-specificity. These review shed light on the multiple possibilities that offer the avian egg model to study the role of serpins in reproduction and developmental biology., (Copyright © 2016 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

To better appreciate the dynamics of yolk proteins during embryonic development, we analyzed the protein quantitative changes occurring in the yolk plasma at the day of lay and after 12 days of incubation, by comparing unfertilized and fertilized chicken eggs. Of the 127 identified proteins, 69 showed relative abundance differences among conditions. Alpha-fetoprotein and two uncharacterized proteins (F1NHB8 and F1NMM2) were identified for the first time in the egg. After 12 days of incubation, five proteins (vitronectin, 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.

Gallin is a 41-residue protein, first identified as a minor component of hen egg white and found to be antimicrobial against Escherichia coli. Gallin may participate in the protection of the embryo during its development in the egg. Its sequence is related to antimicrobial β-defensin peptides. In the present study, gallin was chemically synthesized 1) to further investigate its antimicrobial spectrum and 2) to solve its three-dimensional NMR structure and thus gain insight into structure-function relationships, a prerequisite to understanding its mode(s) of action. Antibacterial assays confirmed that gallin was active against Escherichia coli, but no additional antibacterial activity was observed against the other Gram-positive or Gram-negative bacteria tested. The three-dimensional structure of gallin, which is the first ovodefensin structure to have been solved to date, displays a new five-stranded arrangement. The gallin three-dimensional fold contains the three-stranded antiparallel β-sheet and the disulfide bridge array typical of vertebrate β-defensins. Gallin can therefore be unambiguously classified as a β-defensin. However, an additional short two-stranded β-sheet reveals that gallin and presumably the other ovodefensins form a new structural subfamily of β-defensins. Moreover, gallin and the other ovodefensins calculated by homology modeling exhibit atypical hydrophobic surface properties, compared with the already known vertebrate β-defensins. These specific structural features of gallin might be related to its restricted activity against E. coli and/or to other yet unknown functions. This work provides initial understanding of a critical sequence-structure-function relationship for the ovodefensin family.

Ovalbumin family contains three proteins with high sequence similarity: ovalbumin, ovalbumin-related protein Y (OVAY), and ovalbumin-related protein X (OVAX). Ovalbumin is the major egg white protein with still undefined function, whereas the biological activity of OVAX and OVAY has not yet been explored. Similar to ovalbumin and OVAY, OVAX belongs to the ovalbumin serine protease inhibitor family (ov-serpin). We show that OVAX is specifically expressed by the magnum tissue, which is responsible for egg white formation. OVAX is also the main heparin-binding protein of egg white. This glycoprotein with a predicted reactive site at Lys(367)-His(368) is not able to inhibit trypsin, plasmin, or cathepsin G with or without heparin as a cofactor. Secondary structure of OVAX is similar to that of ovalbumin, but the three-dimensional model of OVAX reveals the presence of a cluster of exposed positive charges, which potentially explains the affinity of this ov-serpin for heparin, as opposed to ovalbumin. Interestingly, OVAX, unlike ovalbumin, displays antibacterial activities against both Listeria monocytogenes and Salmonella enterica sv. Enteritidis. These properties partly involve heparin-binding site(s) of the molecule as the presence of heparin reverses its anti-Salmonella but not its anti-Listeria potential. Altogether, these results suggest that OVAX and ovalbumin, although highly similar in sequence, have peculiar sequential and/or structural features that are likely to impact their respective biological functions.

Background: Most egg yolk precursors are synthesized by the liver, secreted into the blood and transferred into oocytes, to provide nutrients and bioactive molecules for the avian embryo. Three hundred and sixteen distinct proteins have been identified in egg yolk. These include 37 proteases and antiproteases, which are likely to play a role in the formation of the yolk (vitellogenesis), as regulators of protein metabolism. We used a transcriptomic approach to define the protease and antiprotease genes specifically expressed in the hen liver in relation to vitellogenesis by comparing sexually mature and pre-laying chickens showing different steroid milieu., Results: Using a 20 K chicken oligoarray, a total of 582 genes were shown to be over-expressed in the liver of sexually mature hens (1.2 to 67 fold-differences). Eight of the top ten over-expressed genes are known components of the egg yolk or perivitelline membrane. This list of 582 genes contains 12 proteases and 3 antiproteases. We found that "uncharacterized protein LOC419301/similar to porin" (GeneID:419301), an antiprotease and "cathepsin E-A-like/similar to nothepsin" (GeneID:417848), a protease, were the only over-expressed candidates (21-fold and 35-fold difference, respectively) that are present in the egg yolk. Additionally, we showed the 4-fold over-expression of "ovochymase-2/similar to oviductin" (GeneID:769290), a vitelline membrane-specific protease., Conclusions: Our approach revealed that three proteases and antiproteases are likely to participate in the formation of the yolk. The role of the other 12 proteases and antiproteases which are over-expressed in our model remains unclear. At least 1/3 of proteases and antiproteases identified in egg yolk and vitelline membrane proteomes are expressed similarly in the liver regardless of the maturity of hens, and have been initially identified as regulators of haemostasis and inflammatory events. The lack of effect of sex steroids on these genes expressed in the liver but the products of which are found in the yolk suggests that these may be passively incorporated into the yolk rather than actively produced for that purpose. These results raise the question of the biological significance of egg yolk proteases and antiproteases, and more generally of all minor proteins that have been identified in egg yolk.

The chicken egg possesses physical and chemical barriers to protect the embryo from pathogens. OCX-36 (ovocalyxin-36) was suggested to be a 36 kDa eggshell-specific protein that is secreted by the regions of the oviduct responsible for eggshell formation. Its expression is strongly up-regulated during shell calcification. This protein was also detected in vitelline membrane and expressed in gut tissues. Analysis of the OCX-36 protein sequence revealed that OCX-36 is related to the BPI (bactericidal permeability-increasing proteins)/LBP [LPS (lipopolysaccharide)-binding proteins]/PLUNC (palate, lung and nasal epithelium clone) superfamily, and that there are strong similarities between the exon/intron organization of the mammalian LBP/BPI and the avian OCX-36 genes. A recent study revealed that OCX-36 originates from a tandem duplication of an ancestral BPI/LBP/PLUNC gene, after the divergence of birds and mammals. Its antimicrobial activity was recently investigated and it was shown that OCX-36 binds to LPS from Escherichia coli. High-throughput methodologies have led to the identification of approximately 1000 new egg proteins. Among these are LBP/BPI proteins that might play a role in the natural defences of the egg to protect the embryo during its development in the external milieu, and may function to keep the table egg free of pathogens. The function of these BPI-like molecules is the subject of intense research to characterize their putative LPS-binding properties and antimicrobial activity.

Background: As uricoletic animals, chickens produce cleidoic eggs, which are self-contained bacteria-resistant biological packages for extra-uterine development of the chick embryo. The eggshell constitutes a natural physical barrier against bacterial penetration if it forms correctly and remains intact. The eggshell's remarkable mechanical properties are due to interactions among mineral components and the organic matrix proteins. The purpose of our study was to identify novel eggshell proteins by examining the transcriptome of the uterus during calcification of the eggshell. An extensive bioinformatic analysis on genes over-expressed in the uterus allowed us to identify novel eggshell proteins that contribute to the egg's natural defenses., Results: Our 14 K Del-Mar Chicken Integrated Systems microarray was used for transcriptional profiling in the hen's uterus during eggshell deposition. A total of 605 transcripts were over-expressed in the uterus compared with the magnum or white isthmus across a wide range of abundance (1.1- to 79.4-fold difference). The 605 highly-expressed uterine transcripts correspond to 469 unique genes, which encode 437 different proteins. Gene Ontology (GO) analysis was used for interpretation of protein function. The most over-represented GO terms are related to genes encoding ion transport proteins, which provide eggshell mineral precursors. Signal peptide sequence was found for 54 putative proteins secreted by the uterus during eggshell formation. Many functional proteins are involved in calcium binding or biomineralization--prerequisites for interacting with the mineral phase during eggshell fabrication. While another large group of proteins could be involved in proper folding of the eggshell matrix. Many secreted uterine proteins possess antibacterial properties, which would protect the egg against microbial invasion. A final group includes proteases and protease inhibitors that regulate protein activity in the acellular uterine fluid where eggshell formation takes place., Conclusions: Our original study provides the first detailed description of the chicken uterus transcriptome during formation of the eggshell. We have discovered a cache of about 600 functional genes and identified a large number of encoded proteins secreted into uterine fluid for fabrication of the eggshell and chemical protection of the egg. Some of these uterine genes could prove useful as biological markers for genetic improvement of phenotypic traits (i.e., egg and eggshell quality).

Using zymography and mass spectrometry, we identified for the first time the precursor of chicken matrix metalloprotease 2 (pro-MMP-2) as a complex with TIMP-2 (tissue inhibitor of metalloproteinases) in egg white and yolk. Real-time polymerase chain reaction confirmed that MMP-2 and its inhibitors TIMP-2 and TIMP-3 were expressed all along the oviduct and in the liver of laying hens. We also demonstrated that the processing of pro-MMP-2 into mature MMP-2 by serine proteases does not occur in vivo, although purified pro-MMP-2 undergoes proteolytic maturation by these proteases in vitro. Moreover, the relative pro-MMP-2 activity assessed by gelatin zymography was shown to decrease in egg white during the storage of unfertilized or fertilized eggs. However, the mature form of 62 kDa MMP-2 could not be detected. The fact that MMP-2 is found as a proform in fresh eggs suggests that the activity of this metalloprotease is regulated under specific conditions during embryonic development.

Beta‐microseminoproteins (MSMBs) are small disulfide‐rich proteins that are conserved among vertebrates. These proteins exhibit diverse biological activities and were mainly reported to play a role in male fertility, immunity, and embryogenesis. In this work, we focused on the chicken MSMB3 protein that was previously depicted as an egg antibacterial protein. We report that MSMB3 protein is exclusively expressed in the reproductive tissues of laying hens (in contrast to chicken MSMB1 and MSMB2 paralogs), to be incorporated in the egg white during the process of egg formation. We also showed that chicken MSMB3 possesses highly conserved orthologs in bird species, including Neognathae and Palaeognathae. Chicken MSMB3 was purified from egg white using heparin affinity chromatography and was analyzed by top‐down and bottom‐up proteomics. Several proteoforms could be characterized, and a homodimer was further evidenced by NMR spectroscopy. The X‐ray structure of chicken MSMB3 was solved for the first time, revealing that this protein adopts a novel dimeric arrangement. The highly cationic MSMB3 protein exhibits a distinct electrostatic distribution compared with chicken MSMB1 and MSMB2 structural models, and with published mammalian MSMB structures. The specific incorporation of MSMB3 paralog in the egg, and its phylogenetic conservation in birds together with its peculiar homodimer arrangement and physicochemical properties, suggests that the MSMB3 protein has evolved to play a critical role during the embryonic development of avian species. These new data are likely to stimulate research to elucidate the structure/function relationships of MSMB paralogs and orthologs in the animal kingdom. [ABSTRACT FROM AUTHOR]

Chicken diet essentially relies on soybean as the major source of proteins but there are increasing efforts to identify other protein-rich feedstuffs. Of these, some pea cultivars constitute interesting sources of proteins, although some of them contain antinutritional factors that may compromise the digestibility of their protein content. Consequently, chickens exhibit low performance, while undigested compounds rejected in feces have a negative environmental impact. In this article, we analyzed the intestinal content of chickens fed a pea diet (Pisum sativum) to decipher the mechanisms that could explain such a low digestibility. Using gelatin zymography, we observed that the contents of chicken fed the pea diet exhibit altered proteolytic activities compared with intestinal contents from chickens fed a rapeseed, corn, or soybean diet. This pea-specific profile parallels the presence of a 34 kDa protein band that resists proteolysis during the digestion process. Using mass spectrometry analysis, we demonstrated that this band contains the pea-derived Bowman-Birk protease inhibitor (BBI) and 3 chicken proteases, the well-known chymotrypsinogen 2-like (CTRB2) and trypsin II-P39 (PRSS2), and the yet uncharacterized trypsin I-P38 (PRSS3). All 3 proteases are assumed to be protease targets of BBI. Molecular modeling of the interaction of pea BBI with PRSS2 and PRSS3 trypsins reveals that electrostatic features of PRSS3 may favor the formation of a BBI-PRSS3 complex at physiological pH. We hypothesize that PRSS3 is specifically expressed and secreted in the intestinal lumen to form a complex with BBI, thereby limiting its inhibitory effects on PRSS2 and chymotrypsinogen 2-like proteases. These data clearly demonstrate that in chickens, feedstuff containing active pea BBI affects intestinal proteolytic activities. Further studies on the effects of BBI on the expression of PRSS3 by digestive segments will be useful to better appreciate the impact of pea on intestine physiology and function. From these results, we suggest that PRSS3 protease may represent an interesting biomarker of digestive disorders in chickens, similar to human PRSS3 that has been associated with gut pathologies. [ABSTRACT FROM AUTHOR]