DIGE Analysis And Screening Of Differential Proteins In Androgen-induced Antlerogenic Periosteumin Early Period In Female Sika Deer

Deer antlers are male secondary sexual characters,they are the only mammalian appendages capable of full renewal.Antler development refers to the forming process of male sika deer calves pedicle and first antler after puberty.It has confirmed that the piece of periosteum overlying the lateral crest of prepubertal deer frontal bone is a foundation of antler development,which is known as antlerogenic periosteum(AP).AP holds the patterning information for antler formation.Studies suggest that androgens play an important role in antler development,only stimulated by androgen,could AP developed into pedicle and first antler.Li and other researchers found that deer castrated could not develop pedicle,let alone antler.Castration abrogates future pedicle and antler formation.If castrated deers were administrated of exogenous androgens(EA),antler will be induced.In meantime female deer also have the potential to develop pedicles and antlers,but they do not normally express this phenotype due to lack of sufficient androgen stimulation.Suggests indicated that androgen is the prerequisite to the antler development.Interestingly,transformation from a pedicle to an antler seems to be independent of androgen hormones.Antler generation closely associated with the level of testosterone concentration.In order to explore differential expression information of proteins during antler development,the study applied Different Gel Electrophoresis,DIGE to screen for the proteins associated antler developed.Further study the proteomics of periosteum will provide foundation for revealing molecular mechanism of antler development.We carried out experiments were as follows:(1)select two female deer calves in good health,experimental group injected exogenous androgen testosterone(testosterone undecanoate)in vitro,in contrast,control group injected tea tree oil which used to dissolve androgen,both for every other week.A month later detect whether antler pedicle formation or not with pedicle detector,(2)we extract total proteins from the treated and control group's antler periosteum and applied DIGE to obtain the differential expression information of proteins.After that,differentially expressed proteins were identified by Matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry,MALDI-TOF/MS.We summarize the identification results and analysis the information of the differentially expressed proteins through several bioinformatics tools.(3)screening proteins in the results,selected 14-3-3 protein and cloning the gene,furthermore predicted and analyzed its structure through bioinformatics tools.Results were as follows:(1)the experimental deer calf had antler periosteum developed,furthermore,AP cell layer thickened obviously,on the contrary,the control group had not developed.(2)the number of different expression membrane proteins with statistical significance(p<0.05,fold changes?1.5)is 318 by 2D DIGE analyze,163 of them are up-regulated in developed AP and 155 of them are down-regulated.We identify 17 proteins,the proteins were classified into 5 categories with different structures and functions,these proteins are associated with binding,enzyme activity,catalytic activity,antioxidant activity.(3)the Sika deer's YWHAE CDS consisted of 768 nucleotides that encoded 255 amino acids residues,and the amino acid homology of bovine YWHAE shared 99.6% identity with chicken.The analysis of amino acid sequence revealed that the sika deer YWHAE encoded 14-3-3 protein and its relative molecular weight was 29163.86 Da.Subcellular location of 14-3-3 protein was primarily in the cytoplasm,and it belongs to the soluble protein.It is predicted that the 14-3-3 protein contained 19 phosphorylation sites,0 glycosylation sites.The secondary structure of the protein was mainly composed of helix,while the tertiary structure of domain area showed a helical state.Conclusions as follows:(1)female sika deer as well as male has a histological basis of initiation of pedicle growth and antler,androgens play a trophic role in antler growth,artificial induction of antlerogenic periosteum development in female calves which offer the most pertinent model for studying mechanism of antler growth regulation,(2)we get the greatly different patterns of sika deer AP proteins,10 of them are down-regulated in developed AP and 7 of them are UP-regulated,and they are thought involving in antler development,these proteins are likely to the key proteins and give rise to antler generation.14-3-3?,GSN,COL6A2 and ERBBFI1 are closely related to antler development,(3)14-3-3? is a key protein for regulating antler regeneration by effecting the cell migration and cell stability.We cloned and analyzed the Sika deer's YWHAE coding domain sequence(CDS)by RT-PCR,the test confirm the high conservation of the sika deer YWHAE gene expressed 14-3-3 protein.