Cadmium-exposed During Pregnancy Inhibition On F1 Rats' Testosterone Synthesis Through Affecting Steroidogenic Factor 1(SF-1) Expression And Epigenetic Modification

Part ? Role and epigenetic mechanism of steroidogenic factor 1?SF-1?in F1rats'testosterone synthesis inhibition after cadmium exposure during pregnancy.Object:To investigate the effects of cadmium exposure during pregnancy on testicular development and steroidogenesis in F1 rats,role and epigenetic mechanism of SF-1 regulating the expression of steroid hormone synthesis-related factors in F1 rats'of testosterone synthesis inhibition after cadmium exposure during pregnancy.Method:Forty adult SD pregnant rats,randomly divided into 4 groups,starting from the first day of conception,were treated with intragastric administration of saline and cadmium?0.5,2,8 mg/kg CdCl₂?until delivery.F1 male rats were routinely breast-fed and fed to 21 days and 56 days?PND 21 and PND 56?and then sacrificed to gain testis sample and serum.For histopathological examination,after H&E staining,the seminiferous tubule?ST?diameter measurement and Johnsen score were used to evaluate testicular development.Transmission electron microscopy was used to observe testicular ultrastructure.The levels of gonadotrophin releasing hormone?GnRH?,luteinizing hormone?LH?,progesterone?P₄?and testosterone?T?in serum were detected by ELISA.The mRNA and protein levels of SF-1 and steroid hormone synthesis related factors?StAR,CYP11A1,HSD3?,CYP17A1 and HSD17?3?were detected by qRT-PCR and Western blot.BSP was used to detect DNA methylation level in the promoter region of CYP11A1 and CYP17A1 genes;dual luciferase reporter gene was used to verify the binding of SF-1 and StAR to their target microRNAs,respectively,qRT-PCR was used to detect the expression of target microRNAs?miR-10b-5p and miR-328a-5p?related to the regulation of SF-1 and StAR.Results:1 Effects of cadmium exposure during pregnancy on testicular development and serum hormone level in F1 rats1.1 After H&E staining,the diameter measurement showed that the testicular tissue was damaged in both periods.The diameter of the testicular ST increased significantly at PND 21 and decreased significantly at PND 56.The Johnsen score of testicular ST in F1rats after cadmium exposure during pregnancy was significantly reduced,and spermatogenesis was delayed.1.2 In the ultrastructure of testicular tissue of F1 rats in both periods,there were necrotic signs such as sperm chromatin aggregation,cytoplasmic nucleus shrinkage,and intracellular vacuoles and cell gap enlargement.1.3 The levels of GnRH,LH,T and P₄ in the F1 male rats'serum were significantly down-regulated in both periods.2 Effects of cadmium exposure during pregnancy on the expression of SF-1,steroid hormone synthesis related factors in F1 rats and the epigenetic mechanism2.1 After cadmium exposure during pregnancy,at PND 56,the mRNA and protein expression of some steroid hormone synthesis-related factors?StAR,CYP11A1,HSD3?and CYP17A1?in testis were significantly down-regulated in F1 rats.At PND 56,the mRNA expression of some steroid hormone synthesis-related factors?CYP11A1,HSD3?and HSD17?3?increased significantly,but the protein expression was down-regulated.2.2 The mRNA and protein expression of SF-1 in F1 rats were significantly decreased at PND 21,while at PND 56,the mRNA expression was up-regulated,the protein expression was significantly decreased,conversely.2.3 After cadmium exposure during pregnancy,there was no significant change in the total methylation rate of the CpG islands in the promoter region of CYP11A1/CYP17A1 gene in the F1 rats'testes of the two periods.2.4 The dual luciferase reporter gene verified miR-10b-5p and miR-328a-5p were binding to StAR and SF-1 gene,respectively.The expression of miR-10b-5p and miR-328a-5p were changed in F1 rats'testes of the two periods after cadmium exposure during pregnancy.Part ? Role of SF-1 in cadmium-exposed inhibit testosterone synthesis of rat testicular Leydig cells by and the mechanismObject:In the first part,the in vivo study has been observed that SF-1 played an important role in the testosterone synthesis inhibited by cadmium exposure during pregnancy.In the in vitro cell level,further to clarify the role of SF-1 in testosterone synthesis inhibition in rat testicular Leydig cells by cadmium exposure and the epigenetic mechanism.Method:The testicular Leydig cells of 56-day-old SD male rats were extracted,and the cell purity was determined by HSD3?staining,the cell survival rate was detected by blue-staining with different time.After the different doses of cadmium exposure,the cell viability was detected by the CCK8 method at different exposure time to choose the right cadmium exposure time.Apoptosis was detected by Annexin V-FITC/PI double staining and flow cytometry after 48 h cadmium exposure;CCK8 and RT-PCR were used to determine the dose of SF-1 inverse agonist?AC45594,4-heptyloxyphenol?.The P₄ and T levels were detected after 48 h of cell exposure with cadmium chloride alone and in combination with AC45594.QRT-PCR and western blot were used to detect the mRNA and protein expression levels of SF-1 and steroid hormone synthesis related factors.QRT-PCR was used to detect the expression of microRNAs associated with the regulation of SF-1 and StAR gene expression.Results:1 With the increase of cadmium dose and exposure time,the cell viability decreased first,then increased,and the apoptosis rate increased after 48 h exposure.2 The level of P₄ synthesized in testicular Leydig cells was significantly increased and T levels were significantly down-regulated under combined exposure to cadmium chloride and AC45594.3 The mRNA and protein expressions of SF-1,CYP11A1 and CYP17A1 were significantly decreased under the exposure with cadmium chloride alone and in combination with AC45594,respectively.Compared with the control group,the mRNA and protein expression of the three factors showed a more significant decrease in the combined exposure group.4 Epigenetic observed the expression levels of target microRNAs regulated by SF-1and StAR genes?rno-miR-10b-5p and rno-miR-328a-5p,respectively?increased significantly in AC45594 alone exposed group and combined of cadmium chloride and AC45594 exposed group and there was a more significant increase in the combined group.Conclusion:1 Cadmium exposure during pregnancy leaded to impaired testicular tissue,which affected testicular growth and development in the F1 generation weaning period and adulthood;meanwhile,the secretion of gonadal axis hormone?especially T?were inhibited,serum hormone levels were different in the different periods of F1 rats.2 Abnormal expression of the steroid hormone synthesis related factors may be involved in the testosterone inhibition of F1 rats after cadmium exposure during pregnancy.SF-1 may regulate the expression of these steroid-related genes and as an important target for F1 rats'testosterone synthesis inhibited by cadmium exposure during pregnancy.Epigenetic found that miR-10b-5p and miR-328a-5p have a targeting relationship with StAR and SF-1 genes,respectively.After exposure to cadmium during pregnancy,two microRNAs may play a role in post-transcriptional regulation of StAR and SF-1 genes expression in F1 testis,whereas DNA methylation did not play a role in the mRNA expression changes of CYP11A1/CYP17A1 gene.3 At the in vitro level,it is further verified that cadmium up-regulated the expression of SF-1 target microRNA?miR-328a-5p?to reduce the expression of SF-1 in rat testicular Leydig cells,thereby affected the expression of steroid hormone synthesis related factors?CYP11A1,CYP17A1?,and finally inhibited the synthesis of testosterone in cells.SF-1 is an important target for cadmium to inhibit testosterone synthesis in testicular Leydig cells.