VDR Regulating Mouse Cyp11a1 Expression And Androgen Synthesis In Mouse Leydig Cells

CYP11A1 is cholesterol side-chain cleavage enzyme（CYPXIA1）which belongs to the XIA subfamily of the cytochrome P450 family.CYPXIA1 is located in the inner membrane of mitochondria.Its main molecular function is to catalyze the side chain cleavage reaction between cholesterol and pregnenolone（precursor of most steroid hormones）.Therefore,Cyp11a1 is highly expressed in mammalian adrenals,testes,ovaries,uterus and other tissues.CYP11A1 is a key enzyme which involved in the androgen synthesis pathway.Leydig cells are the main place where testosterone is secreted.Vitamin D Receptor（VDR）is a nuclear transcription factor that belongs to the steroid hormone/thyroid hormone receptor superfamily.VDR is widely expressed in various germ cells,such as testicular Leydig cells,support cells,and sperm,indicating it may play a role in male reproduction.Our previous research found that the loss-of-function VDR would reduce the expression level of Cyp11a1gene and the fertility of male mouse.We hypothesized that the VDR as a nuclear transcription factor may regulate the expression levels of Cyp11a1 and the related genes involved in androgen synthesis,and affect the synthesis and secretion of androgen and male mouse reproductive ability.A combination of transcriptome and proteome analyses was used to detect the expression level of Cyp11a1 in wild type（WT）and VDR^-/-mouse,respectively.And the result of Cyp11a1 expression was verified by Western Blot.The Cell immunofluorescence analysis was performed to detect the expression and localization of Cyp11a1.To explore the mechanism of VD/VDR regualating the synthesis of androgen in Leydig cells,TM3 cells were treated with actived VD,VDR interference or overexpression.Finally,we detected the potential VDR-binding elements（VDRE）in the upstream of Cyp11a1promoter region by dual luciferase reporter system.In conclusion,this study revealed that VDR affected estosterone synthesis in male mouse by regulating Cyp11a1 gene expression,which will provide a theoretical basis for study on VDR regulating male reproduction.The main experimental results obtained in this study are as follows:1.The comparative analysis of transcriptome and proteome showed that the expression level of Cyp11a1 was much higher in WT than that of VDR^-/-mouse testis.CYP11A1 was mainly high-expressed in the adrenal gland,testis,but less in heart and skin.Once VDR knockout,the change tendency of CYP11A1 expression was quite different in these tissues.2.Actived VD3 increased expression levels of VDR and Cyp11a1 in mouse TM3 and promoted cell proliferation.Over-expression of VDR can also increase Cyp11a1 expression and promote testosterone synthesis and secretion in TM3 cells.These results showed that Cyp11a1 invloved in the male reproduction via VD/VDR.3.VDR response elements（VDRE）were predicted by bioinformatics software in the gene transcription regulatory region of Cyp11a1.Then,detection of the potential VDRE was carried out by constructing a dual luciferase reporting system.The data implied that VD/VDR regualte the expression of Cyp11a1 directly.