Comparative Yolk Proteomic Analysis Of Fertilized Low And High Cholesterol Eggs And The Impact Of Green Tea Powder On Yolk Proteins Abundance During Embryonic Development

The yolk is the nutritional part of the egg that contains vitamins,minerals,lipids,and proteins which are essential for embryo development and hatching.The egg yolk contains significant amounts of lipoproteins,triacylglycerides,and cholesterol,whose dynamics are indistinct during embryogenesis.The effects of cholesterol on the yolk protein abundance,intensity,and function are ill-defined during embryonic development in chicken.In the present study,using two-dimensional gel electrophoresis,eggs with respective high and low cholesterol protein abundance were investigated after 0,2,6,and 13 days of embryogenesis and further analyzed by matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry.The results revealed that the vitellogenin proteins are the most abundant egg yolk protein that showed proximity and a high degree of variation in isoelectric point and molecular weight.The results demonstrated increased expression of vitellogenin-1 and vitellogenin-3 at two days and vitellogenin-2 protein at 13 days of embryogenesis in both egg types.The ovoinhibitor,immunoglobulin lambda light chain precursor,Ig-gamma(clone-36 chicken),and beta-2-glycoprotein-1 precursor proteins were significantly expressed in high cholesterol eggs while haptoglobin protein PIT-54 and vitelline membrane outer layer proteins intensities were significant in low cholesterol eggs at two days of embryogenesis.The high cholesterol eggs showed a modest increase in egg weight,yolk weight,albumen height,yolk color,and egg shell strength relative to the low cholesterol eggs.The gene ontology enrichment analysis revealed that the differentially expressed proteins such as vitellogenin proteins were involved in lipid transport and lipid localization biological processes and showed nutrient reservoir activity function.The ovotransferrin regulated the biological processes of plasminogen activation and extracellular matrix disassembly and characterized the anchored component of the plasma membrane.The ovoinhibitor protein was involved in response to mineralocorticoid and corticosterone biological processes whereas the vitellin membrane outer layer protein constituted the extracellular exosome,extracellular organelle,and membrane-bounded vesicle cellular components.Furthermore,we added green tea powder(GTP)in chicken feed and observed the changes in low cholesterol and high cholesterol eggs induced by GTP during embryogenesis.The effect of green tea on the egg yolk proteins abundance and the consequent changes in yolk proteins intensities are not well characterized.The GTP induced a modest increase in feed intake,egg weight,yolk weight,color,and albumin height in low cholesterol egg compared to the high cholesterol egg.The GTP supplementation upregulated eight proteins in low cholesterol egg and seven in high cholesterol egg while downregulated four and five proteins in low and high cholesterol eggs respectively.The upregulated proteins were the ovotransferrin,vitellogenin-1,vitellogenin-2,vitelline membrane outer layer,immunoglobulin Y-heavy chain constant region,and immunoglobulin lambda light chain precursor while ovoinhibitor was significantly downregulated in low cholesterol eggs.In high cholesterol eggs,the GTP downregulated most of the yolk proteins including ovotransferrin,vitellogenin-2,vitellogenin-3,ovoinhibitor,and immunoglobulin proteins while upregulated vitellogenin-1 and Ig-gamma(clone-36)-chicken.The majority of differentially expressed proteins constitute the cellular component extracellular space(GO:0005615) and extracellular region(GO:0005576)and regulated the biological processes such as lipid transport(GO:0006869),lipid localization(GO:0010876),positive regulation of plasminogen activation(GO:0010756),negative regulation of substrate adhesiondependent cell spreading(GO:1900025),and iron ion import(GO:0097286).The differentially expressed proteins molecular functions were involved in nutrient reservoir activity(GO:0045735),antigen binding(GO:0003823),potassium channel inhibitor activity(GO:0019870),and lipid transporter activity(GO:0005319).Our results concluded that majority yolk protein abundance altered in high cholesterol and green tea groups that were involved in in vital biological processes and conferred important functions during embryo development while the GTP addition effectively reduced the intensities of ovotransferrin,ovoinhibitors,vitellogenin-2,and vitellogenin-3 in the high cholesterol eggs.Altogether,the finding of this study revealed that the effects of cholesterol content in egg and green tea supplementation on changes in fertilized egg yolk proteins may act through regulating immune function,binding mediation and positive regulation of protein activation.