Abstract
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.
Highlights
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 [1]
The bird amniotic fluid (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
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
Summary
All samples (25 l) 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 Ϫ 20 °C for further analyses by mass spectrometry (supplemental Data S1). 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 g of proteins/well, data not shown) They were obtained as previously described [27]. 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). The phylogenetic analysis and corresponding figures were conducted combining data available in Ensembl databases and in literature
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