| In order to research the changes of proteins profiles and the differences of lipids metabolism in the embryonic broiler chickens liver,a comparative proteomics strategy was applied and AA broiler and Sanhuang broiler with different growth and fat deposition were employed in present study.At the same time,in the comparison of expressions of proteins related to lipids metabolism in two broiler chickens liver,we had sieved the key proteins and enzymes which resulted in the differences of lipids metabolism in two broiler chickens liver.Subsequently,as a physiological regulator,DHEA was injected to AA fertilized eggs before incubation to explore the lipids metabolism-regulating mechanism of it.In order to identify phosphoproteins and investigate their alteration in phosphorylation status associated with DHEA function,AA chicken embryo' liver phosphoproteome was analyzed using 2-DE in combination with western blotting of specific anti-phosphotyrosine.This result of this study provided a new rode for the lipids metabolism-regulating mechanism in broiler chickens.1 Developmental Change of Proteins Profiles in the Embryonic Broiler Chickens LiverThe chicken embryo has been used widely as an experimental model for studying development.The embryonic liver plays a crucial role in metabolism and provides many essential functions.The most common and best-characterized functions include bile production,lipid metabolism,hematopoietic organogenesis and glucose metabolism.In order to research the development pattern,AA broiler and SH broiler were employed in present study.AA chickens are the fat meat-type ones,which have been used to perform the genetic analysis and investigate the development of embryonic chicken liver.SH broiler chicken is a famous Chinese indigenous breed,with uniform fat in body,high intramuscle fat and delicious meat.Proteomic analysis was used to study the molecular mechanism of chicken embryonic liver development.We collected embryonic livers from AA and SH chicken embryos on 9,14,19 days of incubation and hatching.Proteins were extracted and fractionated by 2-DE.Neuhoff's colloidal Coomassie Blue G-250 stain was carried out. Stained gels were scanned and analyzed using PDQuest 7.3 software(Bio-Rad).In-gel trypsin digestion of the differential protein spots,MALDI-TOF-MS was analyzed. Twenty-one and Twenty-six proteins of AA and SH chicken embryonic liver were identified to participate in such processes,including carbohydrate metabolism,cell division, lipid metabolism and signal transduction,especially,two kinds of isozymes of sulfotransferase were detected and their expression patterns exhibited obvious changes among the developmental stages of the liver of broiler chicken embryo.This suggested that sulfotransferase had an important role in the liver function,growth and development of broiler chicken embryo.Our results provide insight into biochemical events taking place during the development of AA and SH chicken embryonic liver.2 Comparison of Expressions of Proteins Related to Lipids Metabolism in Two Broiler Chickens Liver with Different Fat Deposition at Embryonic StageIn order to investigate the difference of fat metabolism during embryonic development, AA broiler and Sanhuang broiler chickens with different growth and fat deposition were employed in present study.They were fed with the same diet and fioor-reared.The liver were gained on days 9,14 and 19 of embryonic development as well as at hatching. Proteins were extracted and fractionated by 2-DE and identified by MALDI-TOF-MS. Peptide mass fingerprinting of the differentially-expressed proteins was performed using the server of MASCOT or Prosperctor or ProFound.Thirty-seven proteins were identified. This study showed that embryonic weight,liver weight increased with development proceeding.Of the identified 37 differential proteins,phosphoenolpyruvate carboxykinase, apolipoprotein A-I,Fatty acid-binding protein(L-FABP), 3-hydroxy-3-methylglutaryl-Coenzyme A synthase were up-regulated in SH chickens.This suggested that the gluconeogenesis,cholesterol metabolism and fatty acids oxidation in the livers of SH chickens were higher than that in the livers of AA chickens.So the differential expression of the L-FABP could have an effect on fat deposition in the chicken.This suggested that some proteins and enzymes of lipids metabolism existed breed character.3 Effect of DHEA on the Expressions of the Proteins Related to Lipids Metabolism in Broiler Chickens Embryo' Liver during Embryonic DevelopmentDehydroepiandrosterone is a precursor of steroid secreted by the adrenal cortex in animal including poultry.In order to explore the possible fat-reducing mechanism of DHEA,this study was to investigate the effect of in ovo administration of DHEA on lipids metabolism and the regulating mechanism of DHEA on lipids metabolism in broiler chickens during embryonic development.The treatment was injected 50μL DMSO cotained 50 mg DHEA per kilogram eggs and the control was only injected 50μL DMSO to fertilized eggs.The livers were gained on days 9,14 and 19 of embryonic development as well as at hatching.The extracted proteins were analyzed using 2-DE.The differential spots were identified by MALDI-TOF-MS.Peptide mass fingerprinting of the differentially-expressed proteins was performed using the server of MASCOT or Prosperctor or ProFound.Thirty-seven proteins were identified.This result showed that embryonic weight and liver weight in DHEA group was significantly lower than that in control group.The expression of apolipoproteinA-I was increased in DHEA group,which facilitated cholesterol to cholesterol esters;the expression of peroxiredoxin 6 which had antioxidative activity was increased in DHEA group;at the same time,sulfotransferase (according to pH5.9) was increased in DHEA group,this demonstrated that DHEA was the best substrate for sulfotransferase(according to pH5.9) in broilers.Above all,the expression of pyruvate dehydrogenase was decreased in DHEA group.This suggested that DHEA reduced the supply of acyl coenzyme A by decreasing the activity of pyruvate dehydrogenase so as to decline the deposition of fat in broiler chicken during embryonic development.4 Study of DHEA on the Expressions of Tyrosine Phosphorylation Proteins Related to Lipids Metabolism in Broiler Chickens Embryo' Liver In order to explore the possible fat-reducing mechanism of DHEA,this study was to investigate the effect of DHEA on lipids metabolism and the regulating mechanism of DHEA on lipids metabolism in broiler chickens during embryonic development.The treatment was injected 50μL DMSO cotained 50 mg DHEA per kilogram eggs and the control was only injected 50μL DMSO to fertilized eggs.The livers were gained on days 9, 14 and 19 of embryonic development as well as at hatching.The extracted proteins were analyzed using 2-DE in combination with Western blotting of specific anti-phosphotyrosine. The differential spots were identified by MALDI-TOF-MS or MALDI-TOF-TOF-MS. Peptide mass fingerprinting of the differentially-expressed proteins was performed using the server of MASCOT or Prosperctor or ProFound.Thirty-two tyrosine phosphorylation proteins were identified.Of which,22 tyrosine phosphorylation proteins showed significantly difference in volume between the control and the treatment.This result showed that DHEA,as the signaling molecule of substance metabolism,had regulated the activity of its target enzyme by the phosphorylation/dephosphorylation;DHEA increased the tyrosine phosphorylation proteins of peroxiredoxin 6 so as to enhanced its antioxidative activity;DHEA increased the tyrosine phosphorylation proteins of sulfotransferase (according to pH5.9) so as to promote the biotransformation of DHEA;what's more, DHEA reduced the supply of acyl coenzyme A by decreasing the activity of pyruvate dehydrogenase so as to decline the deposition of fat in broiler chicken during embryonic development.This could be one of the fat-reducing mechanisms of DHEA.
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