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.

SUMMARYIn 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 to human's. In fact, unlike humans, chicken amniotic fluid does not collect excretory products and its protein composition strikingly changes at mid-development due to 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 non-redundant 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 the 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 betadefensin 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, riboflavinbinding protein). This study provides an integrative overview of the chicken amniotic fluid proteome and opens stimulating perspectives in deciphering the role of the avian egg-specific proteins in embryonic development, including innate immunity. These proteins may constitute Proteomic specificities of the chicken amniotic fluid 3 valuable biomarkers for poultry production to detect hazardous situations (stress, infection, etc.), that may negatively affect the development of the chicken embryo. Proteomic specificities of the chicken amniotic fluid4

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.

The ovalbumin gene family in Gallus gallus is composed of three homologous genes located within a 46 kb locus on chromosome 2: ovalbumin, ovalbumin-related protein Y (OVAY), and ovalbumin-related protein X (OVAX) genes. The expression of these genes in hen oviduct is under estrogen control, but their relative hormonal responsiveness and subsequent protein concentration in egg, is distinctive. Interestingly, all three proteins lack the classical signal peptide for secretion. Ovalbumin, OVAX, and OVAY belong to the serine protease inhibitor (serpin) family whose members share a common tertiary structure. Ovalbumin and OVAX are one of the few members of this family that do not express any protease inhibition activity whereas OVAY has been predicted to be inhibitory, by comparison with the consensus sequence for inhibitory serpins. In contrast to ovalbumin and OVAY, OVAX interacts with heparin, a negatively charged glycosaminoglycan, via a positively charged domain exposed at the surface of the molecule. Ovalbumin is the major egg white protein and might be a source of amino acids for the developing embryo. The physiological function of OVAY is not known, but recent data have revealed a possible role of this protein in early embryonic development. Considering the antibacterial activities of OVAX, this protein might play a role in egg defense. This review sheds light on the expression, biochemistry, and structural specificities of these three highly similar paralogs. It gives new clues in favor of diverging functions, which are likely to have arisen by duplication events from a common ancestral gene.