| Background Estrogen receptor alpha(ERα),a classical nuclear estrogen receptor,which binds with estrogen to mediate gene transcriptional activation and plays an important regulatory role in maintaining the connection function of testicular sertoli cells.A large number of studies have shown that abnormal ERα expression level can lead to male reproductive dysfunction.However,the mechanism of ERα affecting the Blood-testis barrier(BTB)by regulating the testis sertoli cell connection has not been reported.Therefore,this study aims to clarify the regulatory role of ERα on BTB function and further reveal its possible molecular mechanismObjective To investigate the effect of ERα downregulation on the connection function and autophagy level of sertoli cell.The effect of ERα down-regulation on the function of testis Blood-testis barrier(BTB)and autophagy level of mouse testis was studied in vivo.Methods 1.1 CCK8 was used to detect the cell viability of TM4 after 24 h treatment with different concentrations(0,1,5,10,20,50,100,1000 nmol/L)of ICI182780(ICI).The cells were divided into normal control group and different concentrations of ICI(1,5,10,20 nmol /L)treatment group.(1)The number and morphological changes of TM4 cells were observed by optical microscope;(2)The protein levels of ERα was detected by Western blot.The expression of ZO-1,Occludin,Claudin-11,β-Catenin,N-Cadherin,E-Cadherin and Cx43 proteins were detected by Western blot;(3)The expression and localization of Cx43 protein were detected by immunofluorescence;1.2 TM4 cells were treated with different concentrations of ICI for 24 h.(1)The levels of autophagy related proteins LC3 and p62 were detected by Western blot.(2)TM4 cells were divided into normal control group,Chloroquine(CQ,40 μmol/L)group,ICI(20 nmol/L)group and CQ+ICI group.The changes of autophagy related proteins LC3 and p62 were detected by Western blot.The protein changes of ZO-1,β-Catenin and Cx43 were detected by Western blot.2.C57BL/6 male mice aged 7 weeks were purchased.They were randomly divided into normal control group,ICI low-dose group(1 mg/kg),ICI medium-dose group(2 mg/kg)and ICI high-dose group(6 mg/kg),with 13 mice in each group.Subcutaneous injection of 0.05 ml/10 g,once every 3 days.The mice in the normal control group were given equal volume of saline.After 45 days of administration,the mice were weighed and killed after anesthesia.The following indexes were detected:(1)ERα protein expression was detected by Western blot;(2)The weights of testis,epididymis and seminal vesicle were weighed and their indexes were calculated;(3)The epididymis was taken to detect sperm viability and sperm concentration;(4)The morphological changes of testis were observed by HE staining.(5)Biological tracer was used to detect the integrity of the blood-testosterone barrier,3 mice in each group;(6)SOX9-specific labeled sertoli cells were detected by immunofluorescence method,and the number of sertoli cells in testicular tissues of each group was analyzed.(7)Tight junction related proteins ZO-1,Occludin,β-Catenin and Cx43 were detected by Western blot.(8)The expression and localization of ZO-1 and Cx43 in testicular tissue were detected by immunofluorescence.(9)The expression and localization of LC3 B in sertoli cells of testicular tissue were detected by immunofluorescence method.Results 1.1The results of CCK8 showed that the cell viability did not decrease significantly when ICI concentration was below 20 nmol/L.(1)The result of light microscope showed that compared with the normal control group,the cytoplasmic vacuoles increased in ICI group,and the cell morphology had no obvious change;(2)Western blot results showed that the protein expression levels of ERα,β-Catenin and Cx43 in ICI groups were significantly decreased compared with the normal control group.The protein expression levels of ZO-1,Occludin and Claudin-11 of ICI 5 nmol/L and above decreased significantly,while the protein expression levels of N-Cadherin and E-Cadherin did not change significantly.(3)The results of immunofluorescence showed that the fluorescence expression of Cx43 in ICI group decreased significantly,but the position of Cx43 did not change significantly;1.2(1)Western blot results showed that LC3 II was significantly increased and p62 expression was significantly decreased in ICI groups compared with normal control group.(2)Western blot results showed that the expression levels of LC3 II and p62 in CQ+ICI group were significantly increased compared with ICI group.The expression levels of ZO-1,β-Catenin and Cx43 were significantly increased.2.(1)Western blot results showed that ERαexpression in ICI groups decreased significantly compared with normal control group;(2)Compared with the normal control group,the weight and index of testis and epididymis in ICI group decreased significantly,while the weight and index of seminal vesicle increased significantly.(3)Compared with the normal control group,sperm motility in ICI group was significantly decreased,and sperm concentration had no significant change;(4)HE staining results showed that compared with normal control group,the morphology of spermatogenic tubules in ICI low-dose group was basically normal,and no obvious pathological changes were observed.The number of spermatogenic upper cortex decreased,spermatogenic epithelium was disordered and spermatogenic cells fell off in ICI group.The statistical results showed that the thickness of spermatogenic epithelium decreased significantly in the medium and high dose groups of ICI group,and the diameter of spermatogenic tubules did not change significantly in all groups.(5)Biotin tracer experiment showed that compared with normal control group,biotin in ICI medium and high dose groups diffused in the lumen of spermatogenic tubules and showed dose-dependent changes;(6)Immunofluorescence results showed that the number of sertoli cells in ICI group had no significant change compared with the normal control group;(7)Western blot results showed that compared with the normal control group,the expression levels of tight junction related proteins ZO-1 Occludin,occludin and β-Catenin and gap junction protein Cx43 in ICI group were significantly decreased.(6)Immunofluorescence results showed that the fluorescence expression intensity of tight connection-related protein ZO-1 and gap connection-related protein Cx43 in ICI group was significantly decreased compared with the normal control group;(8)Immunofluorescence junction showed that LC3 B expression level of testicular sertoli cells in ICI group was significantly increased compared with normal control group,and fluorescence intensity was gradually enhanced.Conclusion Downregulation of ERα can lead to damage of TM4 cell connection function,and the mechanism may be related to activation of autophagy;Down-regulation of ERα can activate testicular serophagy,leading to damage of testicular serophagy cell connection function and damage of blood-testis barrier function in mice,thus leading to spermatogenic dysfunction. |
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