Identification Of Proteins That Mediate The Role Of Androgens In Antler Regeneration Using Label-free Proteomics In Sika Deer

Background:Deer antlers are the only mammalian appendages that can fully regenerate once lost from their pedicles,the base from which they grow.Antler regeneration is achieved through the recruitment,proliferation and differentiation of the single cell type in the pedicle periosteum（PP）.PP cells are the direct derivatives of the cells resident in the antlerogenic periosteum（AP）,a tissue that exists in prepubertal deer calves and can induce ectopic antler formation when transplanted elsewhere on the deer body.The pedicle periosteum is divided into the potentiated region and the dormant region,which are distinguished by the degree of contacting with the velvet skin.The interactions between the potentiated region and its covering skin prompt antler stem cells to develop into a complete deer antler.Therefore,the proteome of pedicle periosteal tissues may play an important role in the regeneration process.Therefore,antlers offer the most pertinent model for studying organ regeneration in mammals.The previous studies showed that the initiation of antler regeneration by the pedicle periosteum（PP）cells is triggered by the decreasing in circulating testosterone（T）.The level of T has a positive effect on pedicle growth but negative effect on antlers growth.Methods:Blood samples were collected weekly according to the experimental design.Two deer were castrated after first blood sampling to compare the T level before and after regeneration of antler.One deer was administered exogenous T,one was kept control under natural T.Potentiated PP tissues were collected upon antler casting.Androgen hormones were analysed using radioimmunoassay method.The Label-free approach was adopted to detect the differential protein spots.Mass spectrometry carried out to obtain peptide mass finger printing.Signal transduction pathways of identified proteins were explored by using Kobas.3 and string soft wares.Some critical differentially expressed proteins（DEPs）were validated through qRT-PCR.Results:A total of 2956 DEPs of the regeneration over non-regeneration PP tissues were detected.Of these proteins,243 were significantly up-regulated and 98 down-regulated in the regeneration group;2615proteins were detected but not reached significant level in both the regeneration and non-regeneration groups.We identified signal transduction pathways related to androgen hormones,through KEGG（p-<0.05）such as estrogen（E₂）signaling pathway,protein processing in endoplasmic reticulum and focal adhesion.Gene Ontology（GO）annotation of DEPs was classified into Biological process,Cellular components and Molecular functions.Out of all these GO term annotations we selected top ten according to false discovery rate with P<0.05.Discussion:T may have played a key role in deer antler regeneration.Some of the identified DEPs and signaling pathways were found to be related to androgen hormones,which may be involved in antler regeneration.Deer antler regeneration,as a model in biomedical research,is comprehensively explored at all levels（development,cell renewal and repair mechanism）,it is considerably benefit to the humanity in prospects of diseased or damaged human tissues and organs.