| Objective: Idiopathic azoospermia(IA,affecting almost 60% of men with infertility)presents as primary spermatogenesis failure,and its pathogenesis remains being unclear,which has been recognized as one of the most intriguing topics in male infertility.The heterodimeric hypoxia-inducible factors(HIFs)are critical transcriptional regulators of systemic and cellular responses to hypoxia.Some studies reported that the human testis is a naturally O2-deprived organ,and spermatogenesis failure companying with impaired antioxidant capacity was observed in the HIF-?-/-mice.The present study is to investigate how HIF-? is involved in the pathogenesis of spermatogenesis failure in IA patients.Methods: In the present study,we designed a rigorous algorithm and enrolled 30 IA and 30 OA patients respectively.All patients received systemic physical examinations,semen analysis,scrotal ultrasonography,serum hormone tests and testicular sperm aspiration(TESA)according to WHO criteria.We performed H&E and immunohistochemical staining analyses with the TESA specimens to evaluating the abnormal hypoplasia of sertoli cells(SCs)in the IA testes.We further used western blotting analysis to assess the HIF-1?,HIF-2?,superoxide Dismutase(SOD),Hemeoxygenase-1(HO-1),tight junction proteins(ZO-1,Claudin and Occludin),iron importer divalent metal transporter 1 with iron responsive element(DMT1+IRE),iron exporter ferroportin 1(FPN1)and iron regulatory protein1(IRP1)levels in both IA and OA testes.Also,we detected the levels of all the proteins mentioned above in the TM4 cells in vitro after being treated with Bisphenol A(BPA)and nitrogen mustard(NM)respectively.Moreover,the protective effects of the curcumin and N-acetyl-L-cysteine(NAC)for the TM4 cells were evaluated in this study.Results: 1.IA and OA diagnoses were established according to the 2000 WHO Manual for the Standardized Investigation,Diagnosis and Management of the Infertile Male.Totally 220 infertile males were recruited from the Andrology Clinic and Reproductive Medicine Clinic of the Affiliated Hospital of Qingdao University.A rigorous algorithm for the enrollment of IA and OA patients was utilized.Based on the enrolling algorithm,there were ultimately 30 cases enrolled into the IA group and 30 age-matched OA cases into the control group.The age,BMI,and infertility duration at presentation were not statistically different between both the IA and OA groups.This study was conducted with ethical approval from the Institutional Review Board of the Affiliated Hospital of Qingdao University.Written informed consent was obtained from all subjects.2.All of the 30 IA cases had no clear etiologies,and no other diagnoses were applicable.The different etiologies for the OA patients in this study included epididymitis,tuberculosisof the epididymis,CAVD,idiopathic and surgery,etc.3.A systematic physical examination was completed for each patient,with particular focus on the genitalia.The testes of IA patients were palpable and low in the scrotum but notably softer than the OA patients' testes.All of the 30 OA patients' epididymides were visibly distended,and 5 cases showed bilateral absence of the vasa deferentia.According to the scrotal US data,the median volume of the IA patients' testes was even lower than half of that of the OA patients' testes(6.2 ml vs.15.1 ml,P<0.001).4.Follicle-stimulating hormone(FSH),luteinizing hormone(LH),prolactin(PRL)and total testosterone(TT)were determined by electro-chemiluminescent immunoassays in the Clinical Laboratory Center of the Affiliated Hospital of Qingdao University.The median baseline serum FSH and LH concentrations in the IA group were 16.40 IU/L and 10.48 IU/L respectively,and these values were higher than those in the OA group(P<0.001).However,there were no differences in the circulating concentrations of PRL and TT between the IA and the OA groups(P>0.05)5.TESA samples obtained from the IA or the OA patients were fixed in Bouin's solution and embedded in paraffin.H&E stained slides were evaluated by two pathologists independently without knowledge of the patients' clinical features.In the TESA specimens from the IA patients,abnormal SC hyperplasia and shrunken SFT lumens with absent mature spermatozoa were typically observed.Leydig cells were normal.In contrast,in the OA TESA specimens,the epitheliums were cellular during all stages of spermatogenesis,including mature sperm,but lacked the normal ordered arrangement.The central lumens were lost and filled with jumbled desquamated cells.Additionally,the Leydig cells were normal in the OA cases.6.We counted the SC numbers/cross-sections on the H&E stained slides.SC quantification showed that the SC numbers/cross-sections in the IA patients were higher than those in the OA patients(38.0±3.5 vs.19.7±2.6,P<0.001).There were no statistical differences in the cross-section tubule perimeters between the IA and the OA groups(422.15±18.33 ?m vs.433.17 ± 25.61 ?m,P>0.05).The statistical analyses showed that the SCDs in the IA patients were higher than those in the OA patients(0.093 ± 0.008 vs.0.044 ± 0.005,P<0.001).7.Two sequential sections were stained with the SC marker Inhibin ? and the cell proliferation marker Ki-67,respectively.As we expected,the SCs demonstrated strong cytoplasmic Inhibin ? immunoreactivity.The Ki-67 nuclear positive SCs were detected in the IA patients' testes and the Ki-67 LIs in the IA patients were obviously higher than those in the OA patients(48%±6% vs.13%±5%,P<0.001).8.We evaluated the expression levels of superoxide dismutase(SOD)and heme oxygenase-1(HO-1)using western blot analysis.The results showed that SOD levels in the IA patients' testes were decreased(75.2% lower than those in the OA patients' testes,P<0.05),whileHO-1 levels in the IA patients' testes were unaffected.The lack of HO-1 response together with inadequate SOD levels could result in an impaired anti-oxidative capacity in the affected testes.9.HIF-1? and HIF-2? protein levels in both the IA and OA patients' testes were determined by western blotting.The results showed that HIF-1? was dramatically up-regulated in the IA patients' testes(approximately 2.44-fold relative to that in the OA patients' testes,P<0.01).However,HIF-2? protein levels were decreased by 49% in the IA patients' testes compared with those in the OA patients' testes(P<0.01).10.We further determined the expression levels of DMT1+IRE,FPN1 and IRP1 using western blotting.We observed that DMT1+IRE and FPN1 were up-regulated in the IA patients' testes relative to those in the OA patients' testes.DMT1+IRE expression levels in the IA testes were 29% higher than those in the OA testes,indicating enhanced iron import in IA testis(P<0.05).Although there was an increasing trend,FPN1 levels in the IA testes remained being unchanged.Additionally,the protein level of IRP1 was increased by 22% in the IA testes than that in the OA testes(P<0.05).11.In this study,the expression levels of tight junction proteins(ZO-1,Claudin and Occludin)were also determined using western blotting.The results showed that the protein levels of ZO-1 and Claudin in the IA testes were dramatically decreased,only comprising respectively 62.3% and 43.5% of those in the OA testes(P<0.05).The most obvious decrease happened in the expression of Occludin in the IA group,which is only 7.2% of that in the OA group(P<0.05).12.TM4 cells was a sertoli cell line obtained from mouse testis,which was cultured in vitro in this study.TM4 cells were treated with BPA or NM in different concentrations(10-7,10-6,10-5,10-4 and 10-3mol/L respectively).Cell viability was determined by the conventional 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT)assay.When treated with BPA or NM below 10-5mol/L,cell viability was unchanged compared with control.However,a significant reduction in cell viability was observed when TM4 cells were treated with BPA or NM above 10-4mol/L,which were only 57.8% and 24.9%(for BPA treated cells)and 62.2% and 38.6%(for NM treated cells)of control respectively.13.The expression levels of HIF-1? and HIF-2? in the BPA or NM treated TM4 cells were determined by western blotting.In 10-6mol/L BPA or NM treated cells,the up-regulation of HIF-2? was detected(1.41-fold and 1.72-fold relative to those of control respectively,P<0.05);in 10-5mol/L BPA or NM treated cells,on the contrary,the protein levels of HIF-2? were decreased significantly(only 32.2% and 37.7% of those in control respectively,P<0.05).The western blots result of HIF-1? showed that they were up-regulated in 10-6mol/L NM treated cells(2.71-fold compared to control,P<0.05)and down-regulated in 10-5mol/L BPA or NM treated cells(39% and 33.6% of control respectively,P<0.05).While there was only an increased trend of the HIF-1? expression level in the 10-6mol/L BPAtreated cells without any statistically significant difference compare to control.14.Western blotting was applied to determine the protein levels of DMT1+IRE,FPN1 and IRP1 in the BPA or NM treated TM4 cells.The expression levels of DMT1+IRE were significantly higher in the 10-6mol/L and 10-5mol/L BPA or NM treated cells(1.83-fold,1.72-fold,1.75-fold and 1.82-fold compared to control respectively,P<0.05).The same results were also detected for IRP1(1.92-fold,1.5-fold,2.28-fold and 1.87-fold relative to control respectively,P<0.05).However,only an increased trend of the FPN1 level was found in the 10-6mol/L and 10-5mol/L BPA or NM treated cells without any statistically significant difference compare to control.15.There were no statistically significant differences of the HO-1 and SOD protein levels between the BPA/NM treated TM4 cells and the control.16.TM4 cells were treated with curcumin in different concentrations(1,2,5 and 10?mol/L respectively).Cell viability was determined by MTT assay.When treated with the curcumin below 5?mol/L,cell viability was unchanged compared with that of control.However,a significant reduction in cell viability was observed when cells were treated with the 10?mol/L curcumin,which was only 81.9% of control(P<0.05).No protective effects for the BPA treated TM4 cells were observed after incubated with curcumin or N-acetyl-Lcysteine(NAC)respectively.Conclusions: 1.Spermatogenesis failure in seminiferous tubule are observed in the IA TESA specimens.And we verify the abnormal hypoplasia of SCs in the IA patients' testes.The downregulation of SOD protein level was detected in the IA testes,which proves the impaired anti-oxidative capacity in the affected testes.2.We demonstrate that HIF-1? is obviously up-regulated and HIF-2? is significantly downregulated in the IA TESA specimens.Moreover,dose-dependent changes of HIF-1? and HIF-2? protein levels are determined in the BPA or NM treated TM4 cells.Of note,the change of HIF-2? protein level in the BPA or NM treated TM4 cells is consistent with that detected in the IA TESA specimens.3.We also detected the increased expression levels of DMT1+IRE and IRP1 and decreased levels of tight junction proteins in the IA TESA specimens,as well as in the BPA or NM treated TM4 cells.These results might provide new information about the role of HIF-? in the spermatogenesis failure occurred in the IA patients. |
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