Annexin5 Testosterone Synthesis In Leydig Cells Of Rat And The Effect Of Proliferation And Regulatory Mechanism Research

Annexin is a kind of family proteins all of which bind phospholipids in a calcium dependent-manner.160kinds of unique annexins are identified in more than65species from fungi to higher vertebrates. Annexin5is the most abundant member of annexin family, accounting for1%of total cellular protein. Studies show that annexin5is identified as a mediator of the anti-inflammatory activity of glucocorticoids via inhibition of phospholipase A2and an inhibitor of blood coagulation. It also shows protein kinase C inhibitory activity and forms calcium channels on phospholipid membranes. Annexin5is widely distributed in many kinds of tissues, such as the thyroid gland, adrenal gland, testis and other endocrine organs in rat, and its distribution shows cell-specific.Our previous studies have found that gonadotropin-releasing hormone could cause significant increase of annexin5and3β-HSD expression in mRNA level in the rat Leydig cells, and promote the testosterone secretion; other experiments found that annexin5can increase the secretion of testosterone in Leydig cells in a dose and time dependent manner, ERK1/2MAPK was the key signaling pathways regulating the testosterone synthesis by elevating the expression of P450sc、3β-HSD and17β-HSD. Further vivo experiments confirmed that the recombinant rat annexin5can increase testosterone secretion in rats in a dose-dependent manner by elevating the expression of P450scc and3β-HSD. All of obove suggested annexin5may be an important regulatory factor in GnRH regulating reproductive endocrine system.To study the effect of annexin5on reproduction system, we establish The protein profiles of differential expression between the control and experimental group in the process of annexin5stimulate testosterone secretion in cultured rat Leydig cells.The data obtained in this study might be helpful to illuminate the mechanism of annexin5stimulate testosterone secretion in cultured rat Leydig cells.The experiment was divided into four parts. The first part:The primary rat Leydig cells were cultured in vitro and treated with1nmol/L concentration of annexin5for24h. Comparing with the control group, the level of testosterone in the experimental group increased24%(P<0.01). Cell total protein was extracted and the proteins were analyzed by the two-dimensional gel electrophoresis. The differentially expressed proteins were also analyzed by mass spectrometry (MS). After two-dimensional gels electrophoresis,in experimental group, fifty differentially expressed spot were found.From these spots, thirty six proteins were successfully identified by MS.Among of these proteins, twenty-three proteins were found to be overexpressed and thirteen proteins to be underexpressed by mass spectrometry.The second part:to explore the effect of annexin5on the expression of StarD7involved in the process of testosterone production from rat Leydig Cells, The primary rat Leydig cells were cultured in vitro and treated with different doses of annexin5(0.1nmol/L>1nmol/L、10nmol/)for24h to choose the most effective dose of annexin5. Comparing with the control group, the level of testosterone was significantly increased34%(P<0.05) and60%(P<0.01) after treatment with0.1nmol/L and1nmol/L concentration of annexin5respectively, we use RT-PCR and western blotting to detect the expression of StarD7in mRNA level and protein level respectively. Comparing with the control group, in the mRNA level, the expression of StarD7was significantly increased53%and62%after treatment with0.1nmol/L and1nmol/L concentration of annexin5(P<0.01) respectively. In protein level, StarD7expression was significantly increased44%(P<0.05) with1nmol/L concentration of annexin5respectively, testosterone secretion and StarD7expression were significant correlated(r=0.550). we use immunocytochemistry to detect the cellular localization and change of StarD7. Immunocytochemistry showed that StarD7, which was mainly detected in rats testis Leydig cell, had a higher level of expression in the experimental group of1nmol/L annexin5than that of the control group.The third part:to explore the effect of annexin5on the expression of Ect2involved in the process of cell proliferation from rat Leydig Cells The primary rat Leydig cells were cultured in vitro and treated with different doses of annexin5(0.1nmol/L、1nmol、10nmol/L) for24h. we study the effect of annexin5stimulate cell proliferation in rat Leydig Cells and the expression of Ect2. Firstly, we use MTT to detect cell proliferation rate of Leydig cells and flow cytometry to detect phase distribution of cell cycle. MTT assay showed that annexin5promoted the growth of rat Leydig cells in a dose-and time-dependent manner.Flow cytometry analysis revealed that Leydig cells treated with annexin5presented with decreasing percentages of cells in the G2/M phase as time went by. The proportion of cells in the G2/M in cells treated with1nmol/L annexin5for48h and72h decreased from28.39%to24.49%and from34.37%to16.43%respectively (P<0.05). We use RT-PCR and western blotting to detect the expression of Ect2in mRNA level and protein level respectively. Comparing with the control group, in the mRNA level, the expression of Ect2was significantly increased15%and27%after treatment with0.1nmol/L and1nmol/L concentration of annexin5(P<0.01) respectively. In protein level, Ect2expression was significantly increased21%(P<0.05) with1nmol/L concentration of annexin5respectively. Finally, We use RT-PCR to detect the expression of Cell cycle regulatory proteins in mRNA level respectively. The expression of cyclin B1and CDK1increased25%and33%(P<0.05) with1nmol/L annexin5but cdc25c expression had no difference in mRNA level.The forth part:to explore the function and mechanism of annexin5stimulate testosterone production and cell proliferation in rat Leydig Cells. We optimizate transfection conditionion in primary rat Leydig cells, specific silence the expression of annexin5by RNAi, and study the effect of annexin5stimulate testosterone production and cell proliferation in rat Leydig Cells. Compared with control, siRNA showed an inhibitory effect on annexin5in protein level. The protein expression of annexin5significantly decreased by51%(P<0.05) respectively. The level of testosterone was significantly decreased by49%(P<0.01) at24h after silencing the expression of annexin5by siRNA1transfection.The growth of rat Leydig cells was significantly inhibited for2-5days after silencing the expression of annexin5by siRNA1transfection(P<0.01). The proportion of cells in the G2/M for48h significantly increased(P<0.01), while the proportion of cells in the S significantly decreased (P<0.01) respectively after silencing the expression of annexin5by siRNAl transfection. We use western blotting to detect the expression of StarD7and Ect2in protein level respectively. Compared with control, the expression of StarD7and Ect2decreased50%(P<0.01) and28%(P<0.05) with silencing the expression of annexin5by siRNAl transfection.Conclusions:The recombinant rat annexin5could increase testosterone secretion in rat Leydig Cells, annexin5may promote combination of StarD7with cholesterol and mediate cholesterol transported to the inner mitochondrial membrane. Annexin5promote the growth of rat Leydig cells in a dose-and time-dependent manner, and present with decreasing percentages of cells in the G2/M phase as time went by. The mechanism of annexin5playing a protective effect on cell proliferation, one is elevating the expression of cyclinB1and CDK1,another is regulating the expression of Ect2to involve in the mitotic cytoplasm contractile ring formation process.