Isolation, characterization, proteome, miRNAome, and the embryotrophic effects of chicken egg yolk nanovesicles (vitellovesicles)

Abstract

Abstract Egg yolk constitutes about a third of the structure of the chicken egg however, the molecular structure and physiological effects of egg yolk-derived lipid membranous vesicles are not clearly understood. In this study, for the first record, the egg yolk nanovesicles (vitellovesicles, VVs) were isolated, characterized, and used as a supplement for porcine embryo culture. Yolks of ten freshly oviposited eggs were filtered and ultracentrifuged at 100,000 × g for 3 h to obtain a pellet. Cryogenic transmission electron microscopy and nanoparticle tracking analysis of the pellet revealed bilipid membranous vesicles. Protein contents of the pellet were analyzed using tandem mass spectrometry and the miRNA content was also profiled through BGISEQ-500 sequencer. VVs were supplemented with the in vitro culture medium of day-7 hatched parthenogenetic blastocysts. After 2 days of blastocyst culture, the embryonic cell count was increased in VVs supplemented embryos in comparison to the non-supplemented embryos. TUNEL assay showed that apoptotic cells were increased in control groups when compared with the VVs supplemented group. Reduced glutathione was increased by 2.5 folds in the VVs supplemented group while reactive oxygen species were increased by 5.3 folds in control groups. Quantitative PCR analysis showed that VVs significantly increased the expression of lipid metabolism-associated genes (monoglyceride lipase and lipase E), anti-apoptotic gene (BCL2), and superoxide dismutase, while significantly reducing apoptotic gene (BAX). Culturing embryos on Matrigel basement membrane matrix indicated that VVs significantly enhanced embryo attachment and embryonic stem cell outgrowths compared to the non-supplemented group. This considers the first report to characterize the molecular bioactive cargo contents of egg yolk nanovesicles to show their embryotrophic effect on mammalian embryos. This effect might be attributed to the protein and miRNA cargo contents of VVs. VVs can be used for the formulation of in vitro culture medium for mammalian embryos including humans.