Reseach On Bisphenol A Regulation Of Testosterone Synthesis Through Circadian Clock Gene Nr1d1

As an important regulation system,circadian clock plays an important role in regulating various physiological functions ranging from molecules,cells to biochemistry.Studies have shown that the agonist of circadian clock gene Nr1d1 can significantly increase the mRNA expression of StAR with a dose-dependent manner in rat ovarian granulosa cells.Moreover,clock genes are rhythmically expressed in mouse Leydig cells which are involved in regulating the synthesis of testosterone of mice.Bisphenol propane,also known as bisphenol A(BPA),is an important industrial compounds commonly used in the manufacture of daily necessities.As an environmental endocrine disruptor,BPA can get into the body through the respiratory tract,digestive tract,skin and other means.Studies have shown that BPA has potentially toxic effects on the reproductive system of humans and animals and affects the secretion of male testosterone,but the specific molecular mechanism is still unclear and requires further study.In addition,BPA exposure has a significant effect on the level of circadian clock gene expression in neuronal cells.To sum up,we speculate that there should be a certain relationship between BPA,circadian clock and testosterone synthesis.Thus,this study aims to explore the molecular mechanism of BPA on influencing testosterone synthesis through circadian clock.This study mainly used TM3 mouse cell line,primary Leydig cells and mouse testis as the research model.To explore the regulatory mechanism of BPA affecting testosterone synthesis of Leydig cells through circadian clock,various cell biology and molecular biology technologies,such as the Kronos AB-2550 real-time bioluminescence monitoring system,RT-qPCR,Western blotting,ELISA and Flow cytometric analysis,were employed in this study.The results are as following.1.First,an appropriate concentration of BPA treatment(10 ?M)for TM3 cells was screened by CCK8 and Flow cytometric analysis.Then,we used RT-qPCR,Western blotting,ELISA,KronosAB-2550 real-time bioluminescence monitoring system to explore the effects of 10 ?M BPA treatment on the expression of circadian clock genes,testosterone synthesis key enzymes genes and testosterone concentration of TM3 cell and primary Leydig cells.The results showed that 10 ?? BPA treatment changed the circadian clock system of TM3 cells.In addition,the mRNA and protein expression of circadian clock gene Nr1d1 were significantly decreased with BPA exposure.At the same time,the mRNA andprotein expression of key enzyme genes for testosterone synthesis were significantly inhibited,the content of testosterone in the supernatant of TM3 cell culture was also decreased with addition of BPA.The above results indicated that BPA treatment can affect the circadian clock of Leydig cells and the expression of key enzyme genes for testosterone synthesis,thus affecting the synthesis of testosterone from the cellular level.2.In order to detect the effects of BPA on the circadian clock and key enzymes for testosterone synthesis in mice testis in vivo,we successfully constructed a BPA-treated mouse model using peritoneal injection in the dose of 80?M/kg for 7 days.Then we used RT-qPCR,Western blotting and ELISA to detect the changes in mRNA and protein level of circadian clock genes and testosterone synthesis key enzymes genes in BPA-treated testicular tissues and the serum testosterone in BPA-treated mouse.The results showed that the change trend of circadian clock genes,key enzyme genes for testosterone synthesis and serum testosterone content of mice in the BPA treatment group was consistent with the results in BPA treated TM3 cells.Therefore,it was further demonstrated that BPA exposure affect the expression of circadian clock and key enzyme genes for testosterone synthesis in mice and affect the synthesis of testosterone in vivo.3.SR9009,an agonist of clock gene Nr1d1 and Nr1d1 siRNA treatment of TM3 cells was further used to construct the cell models.Kronos AB-2550 real-time bioluminescence monitoring system,RT-qPCR,Western blotting and ELISA were employed to detect the relationship between Nr1d1 and testosterone synthesis of Leydig cells.The results showed that SR9009 caused the disorder of TM3 cell circadian clock system.Moreover,the expression of some key enzyme genes for testosterone synthesis was significantly increased,and the content of testosterone in the supernatant of TM3 cells was significantly increased with SR9009 treatment.On the contrary,the mRNA and protein expression levels of some key enzyme genes for testosterone synthesis in TM3 cells with Nr1d1 knockdown were significantly decreased,and the content of testosterone in the supernatant of TM3 cell culture was significantly decreased.The above results indicate that the circadian clock gene Nr1d1 is positively involved in the regulation of testosterone synthesis in Leydig cells.4.In order to further explore the relationship between BPA,circadian clock gene Nr1d1,and testosterone synthesis,we designed a experiment using TM3 cells with three different treatments,Control,BPA,and BPA + SR9009.Kronos AB-2550 real-time bioluminescence monitoring system,,RT-qPCR,Western Blotting,ELISA and other methods were used to compare the difference.The results showed that treatment with the SR9009 significantly restored the changeing trend of the circadian clock genes,testosterone synthesis key enzyme genes,and the content of supernatant testosterone in TM3 cells caused by BPA treatment.Therefore,the effect of BPA inhibition of testosterone synthesis in TM3 cells may be accomplished through regulating the circadian clock gene Nr1d1 signalling pathway.In sum,our results showed that BPA affects the circadian clock of testis and Leydig cells both in vivo and in vitro,and causes a decrease in the key enzyme genes of the testosterone synthesis pathway at mRNA and protein level.Thus,the synthesis of testosterone is inhibited in Leydig cells with BPA exposure.By exploring the relationship between BPA,circadian clock,and testosterone synthesis,this study indicated that circadian clock gene Nr1d1 is involved in the regulation of testosterone synthesis in Leydig cells,and clarified the molecular mechanism of bisphenol A's influence on testosterone synthesis through regulating circadian clock gene Nr1d1.Our study provides a preliminary theoretical basis for revealing the reproductive toxicity mechanism of BPA and even other environmental estrogens,and solving BPA's damage to male fertility for human beings.