Mechanism Of Leydig Cell Testosterone Synthesis Process StarD7 And Wnt / β-catenin Signaling Pathway Stimulation Annexin5

Annexin5(A5) is a36kDa calcium binding protein containing317amino acids, widely distributed in various cells and tissues. It has been thought to be a kind of ’house-keeping’ protein because its genomic sequences do not contain a TATA box in the5’flanking region. Annexin5has a variety of biological functions, such as the inhibition of protein kinase C, blood coagulation and so on. Our previous studies have found that annexin5regulated the testosterone synthesis in the Leydig cells with a time-and dose-dependent manner.StarD7is a member of the StAR1-related lipid transfer (START) family proteins, which were implicated in lipid transport, metabolism, and signal transduction. START domains, which contained210amino acid residues, bind to specific lipids, including phospholipids, sterols, and sphingolipids. In addition to participation of lipid transport, START domain containing proteins may bind target proteins to membranes and perform certain catalytic functions.Previous studies have shown that twenty-three proteins were found to be over-expressed and thirteen proteins under-expressed by mass spectrometry on the differential expression in the process of testosterone secretion under the stimulation of annexin5in cultured rat Leydig cells. Among these proteins, StarD7was one of the typical raised protein, Moreover, we found that Wnt/β-catenin signaling pathway was involved in the regulation of StarD7.To further investigate the regulation of StarD7and Wnt/β-catenin signal pathway in the process of testosterone production treated with annexin5in the rat Leydig cells, the experiments were divided into three parts.The first part was aimed to study whether StarD7and Wnt/β-catenin signal pathway participated in the testosterone synthesis with annexin5stimulation. Results showed that StarD7, β-catenin were both increased in protein level. The mRNA level of StarD7was also raised, accompanied with the increase of testosterone synthesis. Furthermore, the immunofluorescence results displayed that β-catenin accumulated in the cytoplasm and had the trend into the nuclear of the rat Leydig cells in the experiment groups.The second part validated the influence of testosterone synthesis under the inhibition of Wnt/β-catenin signal pathway on the opposite side, by combination of annexin5and Dkkl, a inhibitor of Wnt/β-catenin signal pathway, to the primary Leydig cells. And preliminary results showed that the testosterone production and StarD7, β-catenin,3β-HSD,17β-HSD expression were all decreased under the treatment with Dkk1alone. Compared with the separate A5group, the production of testosterone and the expressions of StarD7, β-catenin,3β-HSD,17β-HSD were all declined to varying degrees in corporation with Dkkl and annexin5. Hence, it indicated that Wnt/β-catenin signaling pathway was involved in testosterone synthesis through the specific regulation of testosterone synthesis process with StarD7in Leydig cells.The last part, siRNA interference technology was utilized to suppress StarD7protein expression, we investigated the influence of StarD7on the testosterone synthesis in Leydig cells. Firstly, to improve the efficiency of siRNA interference, we conducted a series of conditions optimization, and found that the effect of interference was optimized under the80%cell density,100nmol/L siRNA-StarD7concentration and1:2the ratio between siRNA-StarD7and GenEscortTM Ⅱ, the condition of transfection reagent. On this basis, the testosterone synthesis was decreased significantly after the interference of StarD7expression, along with the decreasd expression of3β-HSD,17β-HSD in protein and mRNA level remarkably. Consequently, the results indicated that the regulation of testosterone production may be indirectly suppressed through StarD7expression on condition of the key enzyme of testosterone synthesis.Thus, we concluded that StarD7and Wnt/β-catenin signaling pathway were both participated in testosterone synthesis under the stimulation of annexin5, the underline mechanism may be explained that the annexin5stimulated the activation of Wnt/β-catenin signaling pathway, then promotes the initiation of StarD7transcription resulting in the expression of key enzyme of testosterone synthesis, eventually leading to an increase of testosterone synthesis.