Study On Abnormal Proliferation And Testosterone Synthesis Of Leydig Cell In Sox30 Gene Knockout Mice

ObjectiveTo investigate the effects and mechanism of Sox30 gene on the proliferation and testosterone secretion of mouse Leydig cells.Methods1.Sox30 gene knockout mice were constructed by gene recombination,and the genotypes of Sox30 wild-type(Sox30^+/+),heterozygous(Sox30^-/+)and homozygous deletion(Sox30^-/-)mice were identified by PCR method.The weight of mice and organs were weighed and the organ index of each organ was calculated.The pathological morphology of testis of mice was observed by HE staining and the levels of testosterone in testicular tissue homogenate were detected by ELISA.2.Lentivirus infection and si RNA interference technology were used to construct Sox30 gene overexpression and knockdown TM3 cell model.CCK-8 method,Ed U staining and Flow cytometry were used to detect cell survival rate and cell cycle distribution.ELISA method was used to detect the levels of testosterone in Sox30 gene overexpression and knockdown TM3 cells.3.On the basis of Sox30 gene overexpression and knockdown cell model,RT-q PCR method and Western blot method were used to detect the m RNA and protein expression levels of cell cycle related genes and testosterone synthesis related molecules in TM3 cells and to explore the molecular mechanism of Sox30 regulating TM3 cell proliferation and testosterone synthesis in vitro.4.In order to further identify the target molecules of Sox30 in regulating the proliferation and testosterone synthesis of Leydig cells in vivo,the testicular tissue of Sox30^+/+,Sox30^-/+and Sox30^-/-mice were used to detect the levels of cell cycle and testosterone synthesis related molecules using RT-q PCR method and immunohistochemical technique.5.Jaspar database was used to predict the binding sites of Sox30 to the promoter region of target genes to explore the regulation mechanism of Sox30 on the expressions of cell cycle and testosterone synthesis related genes.Results1.No significant differences were found on the organ indexes of heart,liver,spleen,lung and kidney among Sox30^+/+,Sox30^-/+and Sox30^-/-mice（P>0.05）.There was no significant difference in testicular organ index between Sox30^+/+mice and Sox30^-/+mice（P>0.05）.Compared with Sox30^+/+and Sox30^-/+mice,the testicular organ index of Sox30^-/-mice significantly decreased（P<0.05）.The interstitial area outside convoluted seminiferous tubule has obvious hyperplasia while the testosterone levels in testis tissue homogenate were significantly lower in Sox30^-/-mice compared with Sox30^+/+and Sox30^-/+mice（P<0.01）.These results suggest that Sox30 is involved in the regulation of Leydig cell proliferation and testosterone secretion.2.In vitro study,we found that the survival rate was significantly lower in Sox30overexpressed TM3 cells than that of the control group（P<0.05）,and the G1 phase cycle arrest was found in Sox30 overexpressed TM3 cells.Besides,the cell survival rate increased and Cell cycle arrest in G1 phase restored obviously after interfering the expression of Sox30gene.At the same time,we found that the overexpression of Sox30 gene can induce testosterone production（P<0.05）,while knockdown the expression of Sox30 can inhibit the secretion of testosterone in TM3 cells（P<0.01）,which is consistent with the results in vivo.3.The m RNA and protein expression levels of Cyclin D1,Cyclin D2,Cdk2 and Cdk6decreased significantly in Sox30 overexpression TM3 cells compared with Vector cells（P<0.05）.On the contrary,the m RNA and protein expression levels of Cyclin D1,Cyclin D2,Cdk2 and Cdk6 increased in si Sox30 group compared with Sox30-NC group（P<0.05）.These results suggest that Cyclin D1,Cyclin D2,Cdk2 and Cdk6 may be the target molecules of Sox30 involved in cell cycle regulation.On the other hand,the levels of Hsd3b1 and Hsd3b2 decreased obviously and the levels of Cyp17a1 and Hsd11b2 increased significantly in Sox30 overexpression TM3 cells（P<0.05）.The opposite phenomenon was observed in Sox30 knockdown TM3 cells.These results suggest that Sox30 may affect the testosterone secretion by regulating the expression of Cyp17a1 and Hsd11b2 in TM3 cells.4.Compared with Sox30^+/+and Sox30^-/+mice,the m RNA expression levels of Cyclin D1,Cyclin D2,Cdk2 and Cdk6 in the testis of Sox30^-/-mice were significantly increased（P<0.01）,which was consistent with the results in vitro.On the other hand,we found that the relative expression level of Hsd3b1 and Hsd3b2 m RNA in testicular tissue of Sox30^-/-mice increased obviously（P<0.05）,but the relative expression level of Hsd11b2decreased significantly（P<0.01）.Immunohistochemical analysis confirmed that Sox30 gene deletion could induce the protein expression of Hsd3b（P<0.05）and inhibit the expression of Cyp17a1 and Hsd11b2（P<0.01）,which was consistent with the results in vitro.5.The predicted results of JASPAR database indicated that there were possible binding sites of Sox30 protein in the promoter regions of Cyclin D1,Cyclin D2,Cdk2,Cdk6,Hsd3b1,Hsd3b2,Cyp17a1 and Hsd11b2,suggesting that the expression of these cycle and testosterone synthesis genes may be transcriptional regulated by Sox30 protein in mice Leydig cells.Conclusions1.Sox30 gene deletion can lead to abnormal proliferation and testosterone synthesis in mouse Leydig cells.2.Cyclin D1,Cyclin D2,Cdk2 and Cdk6 may be the target genes for Sox30 to regulate cell cycle of mouse Leydig cell.3.Cyp17a1 and Hsd11b2 may be the important target genes for Sox30 to regulate testosterone synthesis in mouse.