Vitamin D Receptor(VDR) Regulates Male Reproduction Related Gene Expression And Screening Of Its Interaction Proteins

Vitamin D receptor(VDR),a nuclear transcription factor,was discovered in a large variety of cells and tissues.VDR mainly regulates calcium ion balance,inhibits cell proliferation and stimulates cell maturation and other physiological functions by involving in important signaling pathways such as AKT,Wnt/?-Catenin,NF-?B and so on.Mutations or deletions in VDR could cause dysfunctions in testis,skin,bone,breast,and prostate.In previous studies,we found that VDR knockout male mice had low reproductive capacities and it was speculated that VDR loss may affect the reproductive function of male mice.Therefore,in this study,RNA-seq technology was used to detect gene expression profile in testicular tissue of 6-month-old wild mice and VDR knockout mice.Then,the differentially expressed genes were preliminarily analyzed,and screened out candidate genes related to male reproductive ability.The expression levels of candidate genes were verified by qPCR in vitro.Subsequently,based on yeast two-hybrid technique,VDR interaction proteins were screened from mouse testis cDNA library and their physiological functions were analyzed.Combined with RNA-seq data and yeast two-hybrid screening results,the present study suggested that the molecular mechanism of VDR could affect the reproductive ability of male mouse via direct or indirect actions,which provided basic theoretical data for exploring the physiological functions of VDR.The main results of this study are as follows:1.By comparison,VDR was not detectable in the skin,intestine,kidney and testis of the knockout mice,and the phenomenone indicated that the knockout mice were systemic VDR dysfunction for providing the guarantee for the subsequent performances.The mRNA level of VDR gene in lung,kidney,skin,intestine,testis and thymus was significantly increased after VDR gene knockout in mice,and the expression levels of CYP24A1 gene related to vitamin D metabolism were significantly affected as well.All the data showed that the mice used in this experiment were systemic VDR knockout mice.The knockdown of VDR affected the expression of its target genes,which provided the guarantee for the following experiment.2.Accroding to RNA-Seq data,258 differentially expressed genes were obtained,of which 122 genes were up-regulated and 136 genes were down-regulated.And qPCR verification was carried out to screen for the candidate genes related to reproduction such as KLK1,KLHL4,Angptl4,and Car3,and lipid metabolism related genes Azgp1 andApoF.3.Twelve candidate proteins interacting with VDR were obtained from mouse cDNA library by yeast two-hybrid technique.Among these proteins,AKAP10 and FKBP51 protein may interact with VDR to regulae protein kinase phosphorylation or dephosphorylation,resulting in affecting sperm formation,development and sperm motility.CYP2J5 protein may be associated with VDR to regulate testosterone metabolism.Other protein factors could affect cell proliferation and differentiation,migration and movement,lipid metabolism and other related functions via interacting with VDR.