Investigating proteins and proteases composing amniotic and allantoic fluids during chicken embryonic development

In amniotes, the amniotic fluid is a significant contributor to fetal development and health. While numerous studies have been conducted in mammalian amniotic fluid, the composition of amniotic and other extraembryonic fluids in avian egg along with their physiological functions remain largely unexplored. In such a context, our objective was to characterize the chicken amniotic fluid (AmF) and allantoic fluid (AlF) properties, protein composition, and some associated functions from day 8 to day 16 of incubation. SDS-PAGE combined to mass spectrometry analysis revealed common and specific proteins to each fluid, suggesting distinct properties and functions. Indeed, major AlF proteins are mostly "egg yolk" proteins involved in lipid, vitamin metabolisms, and metal ion transport, while major AmF proteins resemble those of albumen. Drastic changes in the AmF protein profiles were observed during incubation, when the albumen transfers from day 12 onwards, while few changes were detected for the AlF protein profile. The decreases in osmolality (from 231 to 183 mOsm/kg) and pH (from 8.26 to 7.26) observed in the AlF during incubation are associated with water and electrolytes reallocation for the embryo needs. In contrast, AmF pH value remained stable (≈7.5). Active proteolytic enzymes have been identified in the 2 fluids using gelatin zymography, followed by mass spectrometry analysis for protease identification. A total of 12 proteases was detected in the AlF, compared to 5 in the AmF. We have shown that AlF concentrates proteolytic enzymes assumed to participate in digestive processes: aminopeptidase N, dipeptidyl peptidase-4, meprin A, and 72 kDa type IV collagenase preproprotein. The other proteases identified in both fluids also could have a role in morphogenesis (hepatocyte growth factor activator, suppressor of tumorigenicity 14, astacin-like metalloendopeptidase) and hemostasis (prothrombin and coagulation factor X). Altogether, these data suggest that the roles of chicken AlF and AmF are not merely associated with protection of the embryo and regulation of metabolic disposable wastes, but also they could have more sophisticated roles during embryonic development.