Effects Of Dhea On Biological Characteristics And Its Regulating Testosterone Production Mechanism In Primary Rat Leydig Cells

As the important precursor of steroid hormones synthesis in the body, dehydroepiandrosteron (DHEA) is the the most important metabolic intermediate of steroids. It has many biological functions, and playing an important role in the nervous system, immune system, bone metabolism, lipid metabolism and so on. At present, many scholars believe that DHEA exerts its special biological functions due to it could conversion to testosterone or estradiol in the peripheral target tissues/target cell. Leydig cells located in the loose connective tissue of the testis seminiferous tubules, and its main function is to synthesize and secrete testosterone,95%of the plasma testosterone is secreted by Leydig cells. Our previous research results show that exogenous addition of DHEA can increase the content of testosterone and estradiol in serum of different physiological period model in rats. DHEA treatment could significantly inhibit the proliferation of TM-3cells, but it can significantly enhance the mitochondrial function of TM-3cells. Also, it is found that DHEA is transformed to testosterone and estradiol in the TM-3cells, but its mechanism is still not very clear. Therefore, we investigate the effect of DHEA on the biological characteristics of primary rat Leydig cells proliferation, cell cycle, mitochondrial membrane potential, which is based on separation, purification and identification of primary rat Leydig cells; After confirming the DHEA’s effect on testosterone production in Leydig cells, we focus on the impact of DHEA on the expression of steroidogenic enzymes upon the protein level, and then explore the effect of the MAPK-ERK signaling pathway and nuclear transcription factor of CREB in the regulation of primary rat Leydig cells’secreting testosterone. The study aims to reveal the effects of DHEA on specific target cell biological characteristics, and clarify the mechanism of synthetic steroid hormones deeply from the signal transduction pathway, in order to enrich theoretical and experimental basis for elucidating the biological function of DHEA.1Effects of DHEA on cell proliferation characteristics in primary rat Leydig cells In this study, we main explored the effect of DHEA on cell proliferation and cell cycle in rat Leydig cells. The cells were treated with medium including the final concentration0μmol/L,1μmol/L,50μmol/L and100μmol/L DHEA for24h. The effect of DHEA on cell proliferation was observed using inverted phase contrast microscope and EdU method. The cell cycle was measured by flow cytometer for6h,12h,24h and48h administrated with DHEA respectively. The CyclinA, CDK2and CyclinB mRNA expression level in rat Leydig cells were determined after treated24h by DHEA using the Real-time PCR. The results showed that DHEA could significant inhibitory cell proliferation and EdU method further confirmed the high concentration of DHEA could inhibit cell proliferation in rat Leydig cells. Compared with control group, after treated with100μmol/L DHEA for12h and24h, the cell ratio of S phase increased and G2/M phase decreased significantly (P<0.01), and the same results occurred after50μmol/L and100μmol/L DHEA treated at48h. The CyclinA and CDK2mRNA levels significantly decreased with100μmol/L DHEA group compared to the control group (P<0.05), but there was no significant difference in the CyclinB mRNA level (P>0.05). These results suggested that DHEA could inhibit cell proliferation of primary rat Leydig cells through significantly down-regulated the CyclinA and CDK2mRNA expression to remain Leydig cell in S phase.2Effects of DHEA on structure and function of mitochondria in primary rat Leydig cellsIn this study, we mainly explored the effects of DHEA on the structure and function of mitochondria in primary rat Leydig cells. The cells were treated with different concentration of DHEA, and then detect cell viability by MTT method the shape and quantity of mitochondria were investigated by TEM after treated with DHEA, the impacts of DHEA on mitochondria membrane potentials (△ψm) was determined using flow cytometer, and the activity of succinate dehydrogenase (SDH) was measured by assay kit. The results showed that compared with the control group, Leydig cells viability was significantly increased by0.1μmol/L,1μmol/L,10μmol/L,50μmol/L,100μmol/L and200μmol/L DHEA-treatment at24h and48h (P<0.01), and the same results occurred after administration50μmol/L,100μmol/L and200μmol/L DHEA after treated at48h (P<0.01). There was no significant differences in both shape and quantity of mitochondria in rat Leydig cells with1μmol/L,50μmol/L and100μmol/L DHEA-treated (P>0.05). Compared with the control group, the different concentration of DHEA could reduce mitochondria membrane potential of Leydig cells at24h, and100μmol/L DHEA group was significantly decreased (P<0.01). The activities of SDH were significantly increased in both50μmol/L and100μmol/L DHEA group (P<0.01), but there was no significant difference in1μmol/L group (P>0.05). These results indicated DHEA could decline mitochondria membrane potential and increase its permeability, meanwhile increase SDH activity for improving the level of oxidative metabolism, to improve the cell viability in rat Leydig cells.3Effects of DHEA on the synthesis of testosterone and the key steroidogenic enzymes expression in primary rat Leydig cellsIn this study, we aimed to explore the influences of exogenous DHEA treatment on the content of testosterone and the expression of steroidogenic in primary rat Leydig cells.. The primary Leydig cells were treated with medium including the final concentration0μmol/L,1μmol/L,50μmol/L and100μmol/L DHEA for24h, the content of testosterone was detected by the supernatant radioimmunoassay, the mRNA levels of3β-HSD and17β-HSD were detected by Real-time PCR. With0μmol/L and100μmol/L DHEA incubating the primary rat Leydig cells for3h,12h,24h and48h, the protein levels of3P-HSD,17β-HSD and Aromatase were detected by Western blot. The results showed that the content of testosterone could increase significantly by DHEA. Also,100μmol/L DHEA could significantly improve the mRNA level of3β-HSD (P<0.01) and17β-HSD (P<0.05).100μmol/L DHEA treatment cells for12-48h, the protein level of3β-HSD was significantly increased when compared to the control group (P<0.05), the protein level of Aromatase was decreased with100μmol/L DHEA for48h (PO.05), but there was no significant change in protein levels of17β-HSD for different times (P>0.05). Compared with the control group of the same time, the protein level of3β-HSD was significantly increased with100μmol/L DHEA for24h-48h (P<0.05), the protein level of17β-HSD was significantly increased with100μmol/L DHEA for24h-48h (P<0.05), and the protein level of Aromatase was significantly decreased with100μmol/L DHEA for24h-48h (PO.05). These results suggested that DHEA treatment could significantly increased the3β-HSD and17β-HSD expression, decreased of the Aromatase expression, thus contributing to the synthesis of testosterone in primary rat Leydig cells. 4The effect of DHEA on the signal pathway with related to testosterone synthesis in primary rat Leydig cellsIn this study, we selected the primary rat Leydig cells as the research object, and further to explore the effect of exogenous DHEA treatment on testosterone synthesis related signal transduction pathways. The results showed that the protein level of ERK1/2did not show significant change in different concentration of DHEA treatment (P>0.05), but50μmol/L and100μmol/L DHEA treatment could significantly increase the level of p-ERK1/2protein (P<0.01). The level of ERK1/2protein did not change after treated with100μmol/L DHEA for different time, but100μmol/L DHEA-treated for30min and120min could significantly increase the level of p-ERK1/2protein (P<0.01). After adding the inhibitor U0126of p-ERK1/2, the effect of DHEA on p-ERK1/2could be completely reversed. With or without the presence of the inhibitor conditions,1μmol/L,50μmol/L and100μmol/L DHEA treatment could significantly increased the content of testosterone in primary Leydig cells (P<0.01), and showed dose effect obviously. The content of testosterone with inhibitor treatment group was significantly reduced when compared to uninhibitor treatment group in the same doses of DHEA treatment conditions, and50μmol/L and100μmol/L DHEA treatment showed a significant decreased (P<0.05). Compared with the control group,100μmol/L DHEA treatment could significantly improve the level of3(3-HSD and17β-HSD protein in the primary rat Leydig cells (P<0.05), the effect of DHEA on expression of3β-HSD and17(3-HSD protein could be significantly reversed by the inhibitor U0126pretreatment (P<0.05). Also,100μmol/L DHEA treatment could significantly decrease the expression of Aromatase protein (P<0.05), but the effect of DHEA on the Aromatase protein could not be changed under the inhibitor U0126pretreatment (P<0.05). The epression level of CREB protein did not show significant change with100μmol/L DHEA treatment for30min in primary rat Leydig cells (P>0.05). However, the expression level of p-CREB protein was significantly increased with50μmol/L and100μmol/L DHEA treatment for30min. These results suggested that DHEA treatment could regulate the expression of steroidogenic enzymes by actived the MAPK-ERK signaling pathway and increased the protein expression level of p-CREB which resulting in the testosterone synthesis was enhance in primary of rat Leydig cells.