Yolk small extracellular vesicles in oviparous vertebrates: protein profiles of YsEVs from unfertilized and fertilized eggs reveal their potential biological function for early embryo

Small extracellular vesicles (sEVs) exert crucial biological functions in a wide range of organisms, whereas the existence characteristics and biological roles of egg yolk-derived sEVs (YsEVs) in oviparous vertebrates remain largely uncharacterized and unexplored to date. Here, YsEVs were respectively isolated from 13 Aves and Reptilia species via differential centrifugation, and characterized using TEM, NTA, and WB. All YsEVs exhibited typical sEV features with sEVs marker protein patterns across species. Interestingly, nearly all characteristic parameters (diameter, volume) of YsEVs derived from Aves exhibited relative similarity. In contrast, within Reptilia, the diameter of YsEVs derived from Squamata (snakes) was significantly larger than that of other reptilian samples. Proteomic analysis of YsEVs isolated from fertilized (F-YsEVs) and unfertilized (uF-YsEVs) chicken eggs identified 694 proteins in total. Compared with uF-YsEVs, F-YsEVs exhibited 106 up-regulated and 144 down-regulated proteins (|FC| > 1.2 and P < 0.05). Bioinformatics analysis revealed that these differentially expressed proteins mediate critical embryotrophic functions: nutrient/energy supply, developmental regulation, antimicrobial protection, antioxidant activity, and metabolic signaling. Collectively, sEVs are widely present in the egg yolks of oviparous vertebrates. Among the protein cargo of YsEVs, the differentially expressed proteins between F-YsEVs and uF-YsEVs are of particular interest, given that they elucidate the potential Biological Function for early embryo (stage X) in these organisms.