Effects Of Cadmium Exposure During Pregnancy On Sertoli Cells Of The Testis And Relatede Genes DNA Methylation In The Offspring Of Paternal Rats

Part Ⅰ Effects of cadmium exposure during pregnancy on Sertoli cells of offspring ratsObject:The effect of cadmium exposure during pregnancy on the Sertoli cells of the offspring rats.Methods:SPF of SD rats 250±10g,male and female 1:1 or 1:2 caged,checked the vaginal suppository or vaginal smear the next morning,found the vaginal suppository or smear the sperm is recorded as the 0th day of pregnancy.The pregnant rats(F0 generation)were randomly divided into 4 groups,and cadmium chloride(0,0.5,1,2 mg/kg/day)was given by intragastric administration,and the exposure time was from the first day of pregnancy until delivery(ie,1-21 days).The F0 mice gave birth to F1 male mice after natural delivery,and they were fed regularly for 5,21 and56 days(PND5,PND21,PND56),and each group of F1 adult male mice was randomly selected and caged with healthy female mice 1:2 mating produces the F2 generation,and the F2 generation male rats were routinely fed to produce the F3 generation adult male rats in the same way.Collecting F1,F2,and F3 generation serum and testis,performing histopathological examination,measuring the diameter of seminiferous tubule(ST)after H&E staining,and evaluating the growth and development of testis with Johnsen score;observing the cell ultrastructure by transmission electron microscope;ELISA method was used to determine serum levels of Gonadotropin-releasing hormone(Gn RH)and Follicle Stimulating Hormone(FSH);immunohistochemical method was used to detect FSHR localization and expression in testicular tissue cells.Results:1 Effect of cadmium exposure during pregnancy on the growth and development and testicular histopathology of offspring rats1.1 Basic data showed: Compared with the control group,the F1 generation rat’s weight was only at PND5,and the 2mg/kg dose group gained weight(P<0.05);the F2 generation rat’s weight was only at PND21,the 1mg/kg dose group gained weight(P<0.05).1.2 H&E staining showed :The testes of the F1 generation rats were only at PND5,and the diameter of the seminiferous tubules of the 1 mg/kg dose group was reduced(P<0.05);the testes of the F2 generation rats were only at PND56,and the testicular flexure of the 0.5 mg/kg dose group The diameter of the seminiferous tubules decreased(P<0.05),and the diameter of the seminiferous tubules increased at2mg/kg(P<0.05);the diameter of the seminiferous tubules in the testis of F3 generation rats increased by 0.5mg/kg(P<0.05),the Johnsen score of each dose group increased(P<0.05);the light microscope showed that no abnormal changes were seen in the Sertoli cells of the testis at each stage of the three generations,and intact spermatogenesis was seen in the seminiferous tubules.2 Effects of cadmium exposure during pregnancy on the ultrastructure of testicular Sertoli cells of offspringTransmission electron microscopy showed that in F1 rat PND5 and PND56 Sertoli cells in the control group had intact cell membranes,intact nuclear membranes and close contact with surrounding cells.In the dose group,changes in the ultrastructure of testicular Sertoli cells were observed,with obvious vacuolization,edema,and edema.Necrosis,and the gap with surrounding cells increases.3 Effects of cadmium exposure during pregnancy on serum hormones of offspringSerum hormone level test showed: F1 generation result: Gn RH increased at0.5mg/kg in PND21(P<0.05).FSH increased at 0.5 mg/kg at PND5(P<0.05);at PND21,serum FSH levels in each dose group decreased(P<0.05).Results of F2generation: FSH increased at PND5,0.5mg/kg(P<0.05).Results of F3 generation:Gn RH and FSH have no obvious changes in F3 generation.4 Effects of cadmium exposure during pregnancy on the secretion of FSHR protein by Sertoli cells of offspringAfter exposure to cadmium during pregnancy,F1 generation PND56 testis tissue was stained by immunohistochemical staining.Sertoli cells were observed on the basal surface of seminiferous tubules,and no free Sertoli cells were found.FSHR of normal group was distributed on the basal surface,and the protein AOD value was0.5090.The FSHR protein in the group was also distributed on the basal surface of the seminiferous tubules,and the protein AOD value was 0.4011.Compared with the control group,the FSHR protein AOD value was lower(P<0.05).Part Ⅱ The mechanism of cadmium exposure during pregnancy affecting the testicular Sertoli cellsObject:Transgeantional effects of cadmium exposure during pregnancy on Sertoli cells of offspring rats.Methods:Collecting the first part of the testis,using q RT-PCR and Western blot to detect FSHR/PI3K/AKT/FOXO1 pathway related m RNA and protein expression and related DNA methyltransferase(DNMT1,DNMT3 A,DNMT3B)m RNA and protein expression;Agena Mass ARRAY (?) Methylation method detects the methylation level of promoter regions of fshr,akt and foxo1 related genes.Results:1 Effects of cadmium exposure during pregnancy on FSHR/PI3K/AKT/FOXO1 pathway related factors in the testis of offspring rats1.1 Results of F1 generation: Compared with the control group,the expression of fshr m RNA in the 0.5 mg/kg dose group increased in male rats at PND5(P<0.05),and the expression of AKT protein increased in the 1 mg/kg dose group and the FOXO1 protein 2 mg/kg dose group(P<0.05);At PND21,the m RNA expression of foxo1 in the 0.5 mg/kg dose group increased(P<0.05),and the protein expression decreased(P<0.05),and the protein expression of FSHR and AKT increased in the 2mg/kg dose group(P<0.05);At PND56,the m RNA expression of akt in the 1 mg/kg dose group increased(P<0.05),and the expression of FOXO1 protein increased(P<0.05).1.2 Results of F2 generation: Compared with the control group,at PND21,the m RNA expression of fshr in the 0.5 and 1 mg/kg dose groups decreased(P<0.05),PI3 K protein in the 2 mg/kg dose group,and FOXO1 protein expression increased at1 mg/kg(P<0.05).However,the expression of AKT protein decreased in the 1 mg/kg dose group(P<0.05);at PND56,the expression of AKT in the 1,2 mg/kg dose group and the FOXO1 protein expression in the 2 mg/kg dose group increased(P<0.05).1.3 Results of F3 generation: Compared with the control group,when male rats were adults,the m RNA expression of akt in the 1 mg/kg dose group increased(P<0.05),but the m RNA expression of foxo1 in the 2 mg/kg dose group decreased(P<0.05),the protein expression of FSHR in the 1mg/kg the dose group increased(P<0.05).2 Effects of cadmium exposure during pregnancy on DNA methyltransferase in testis of offspring rats2.1 Results of F1 generation: Compared with the control group,when the male rats were at PND5,the m RNA expression of dnmt3 a in the 1 mg/kg dose group decreased(P<0.05),and the m RNA expression of dnmt3 b in the 2 mg/kg dose group increased(P<0.05),while at 2 mg/kg the protein expression of DNMT3 A and DNMT3 B in the dose group increased(P<0.05);at PND21,the m RNA expression of dnmt1 and dnmt3 a in the 0.5 mg/kg dose group increased(P<0.05);at PND56,the m RNA expression of dnmt3 a in the 2 mg/kg dose group increased(P<0.05),and the protein expression of DNMT3 B in the 1,2mg/kg dose group increased(P<0.05).2.2 Results of F2 generation: Compared with the control group,the protein expression of DNMT1 in the 2 mg/kg dose group increased in male rats at PND5(P<0.05);at PND21,the m RNA expression of dnmt3 a in the 1 mg/kg dose group decreased(P<0.05);At PND56,the m RNA expression of dnmt3 a in the 0.5 and 2mg/kg dose groups decreased(P<0.05).2.3 Results of F3 generation: Compared with the control group,in male adult rats,the m RNA expression of dnmt1 in the 1 mg/kg dose group increased(P<0.05),and the protein expression of DNMT1 and DNMT3 B in the 2 mg/kg dose group increased(P<0.05),but the protein expression of DNMT3 A in the 1 and 2mg/kg dose group decreased(P<0.05).3 Effect of cadmium exposure during pregnancy on the methylation level of the promoter region of the FSHR/PI3K/AKT/FOXO1 pathway related factors in the testis of offspring rats3.1 Results of F1 generation: Compared with the control group,the methylation level of the male rats at PND21 at position 65 of foxo1-14 fragment,28 th and 29 th positions of foxo1-17 fragment decreased(P<0.05),At PND56,the methylation level of the dose group at position 2 of the akt-9 fragment decreased(P<0.01).3.2 Results of F2 generation: Compared with the control group,the methylation level of the fshr-8 fragment at the 4th position group of male rats at PND21 decreased(P<0.05),and the akt-33 fragment at the 12 th position group of basement decreased(P<0.05),and the level of methylation at the 21 st and 22 nd dose groups of foxo1-15 fragment increased(P<0.05).Conclusion:1 Cadmium exposure during pregnancy can damage the testicular tissue and supporting cell structure of the offspring,and affect the growth and development of the testis.The changes in each period are not continuous,but have a cross-generation toxic effect;it can interfere with the secretion of multi-generation serum hormones(Gn RH,FSH)and Inhibit the expression of FSHR protein in Sertoli cells in F1 testicular tissue,thereby interfering with the normal function of Sertoli cells in testicular tissue.The possible main mechanism is to regulate the expression of FSHR/PI3K/AKT/FOXO1 pathway related factors that affect the proliferation of supporting cells;2 Cadmium exposure during pregnancy can affect the DNA methylation pattern of testis tissue by interfering with the expression of DNA methyltransferases(DNMT1,DNMT3 A,DNMT3B)at various generations and stages,and has a cross-representative genetic effect;it also changes fshr,akt,and foxo1 gene methylation level of different Cp G sites in the promoter region,but has no effect on the overall gene promoter region methylation level of the three genes.The effect of cadmium exposure on the transcription level of the offspring fshr,akt,and foxo1 may not be through DNA Methylation modification.