The Role Of Retinoic Acid In Remolding Spermatogenesis Process Of Cryptrochidism

Background: Cryptorchidism is one of most common congenital malformations in children,and may cause male infertility.The diagnose of cryptorchidism is based on clinical manifestation,rather than etiology.Although the etiology of cryptorchidism is still ambiguity,it may cause testicular malignant transformation,testicular torsion and male infertility.Orchidopexy is usually performed for cryptorchid treatment,while surgery could not correct damaged germ cells in cryptorchidism testes.It is crucial to investigate the therapeutic strategies to improve spermatogenetic function in cryptrochid.Retinoic acid(RA)is the metabolite of Vitamin A.It has reported that RA signaling pathway is indispensable for normal spermatogenic process.The relation between RA and cryptorchidism have not been described.It is interesting to note that the spermatogenesis-blocking process in cryptorchidism is consistent with the spermatogenesis failure process in the RA-deletion model.We hypothesize RA decrease may become the vital etiology in cryptorchidism.So we first guess that supplement of RA may recover the damaged spermatogenic process in cryptorchid testes,which could provide some novel approach for cryptorchidism treatment.Objective: We plan to establish congenital cryptorchidism rats model,and administrated cryptorchidism rats with RA in spermatogenesis critical period after birth.Combine experiments in vivo and in vitro,to investigate the molecule mechanism of RA on cryptorchidism,which may provide some scientific foundation for investigating of cryptorchidism etiology and cryptorchidism treatment in clinic.Methods: 1.Sprague-Dawley pregnant rats were injected daily with 25 mg/kg/d flutamide at ED12-ED20 to induce cryptorchidism in the male fetuses.The total number of male cubs and the incidence of cryptorchidism were detected.The protein level of SCP3 and Stra8 were used to select proper RA treatment period with 1 mg/kg/d.PND 1-10 d was screened as proper period.The effect of RA on pathology change was evaluated.The key markers of spermatogenic process were detected,including Stra8,SCP3,c-Kit and Mvh.Besides,sperm count and malformation rate were also detected.All above experiments were performed to investigate the effect of RA on spermatogenic process in cryptorchidism testis.2.All male cryptorchidism rats were randomly apportioned to three groups,normal control,treated with flutamide only(Flu-only),and treated with Flu and RA(Flu + RA).The BTB junction proteins(Connexin43,ZO-1,N-cadherin and Claudin11)were detected with Western-blot and immunofluorescence.The permeability function of BTB was detected with Biotin tracer.The results were in vivo also evaluated with testicular culture in vitro.We mainly discussed the effect of RA on BTB in this section.3.The potential pathway in RA-induced stem cell differentiation process was predicted with bioinformatics methods.The key proteins level of PI3K/Akt/m TOR signaling pathway were detected with Western-blot and immunofluorescence.The proteins level of m TOR-relate macroautophagy were also evaluated,including Beclin1,LC3a/b and P62.To investigate the function of RAR? in RA induced spermtogenic process in cryptorchidism testis,RAR? was blocked in testicular culture in vitro.The effect of RA on PI3K/Akt pathway in GC-1 cells and TM4 cells were also detected with Western-blot,respectively.The potential molecular mechanism of RA on spermatogenic process was discussed in this section.Results: 1.Cryptorchidism could be induced with flutamide administrated in pregnant rats.PND 1-10 was selected as proper time period for RA administrated in cryptrochidism offspring rats.There has no significant influence on nervous system function(according Garcia scores)and blood corticosterone(p>0.05).RA could improve pathology damage partly when supplement during spermatogenesis critical period.Compared with normal control group,the proteins level of SCP3,c-Kit and Stra8 were significantly decreased in Flu-only group(p<0.05),while could be improved after supplement with RA in cryptorchidism testis(p<0.05).There was no significantly change of sperm count and sperm deformity rate between normal control group and Flu+RA group(p>0.05).Compared with normal control group,the protein expression of RAR? was significantly decreased in Flu-only group(p<0.05),and could be improved after RA treatment(p>0.05).2.RA supplement could significantly improve proteins expression of Connexin43,ZO-1,N-cadherin and Claudin 11 compared with Flu-only group(p<0.05),Biotin tracer indicated that BTB permeability could be ameliorated after RA supplement in cryptorchidism testis.3.KEGG result showed that PI3K/Akt signaling pathway may have crucial role in RA induce stem cell differentiation process.Compared with normal control group,the proteins expression of PI3 K,p-Akt/pan-Akt and p-m TOR/m TOR were significantly increased in cryptorchidism testis,the level of Beclin1 and LC3 a/b was significantly decreased while p62 was significantly increased(p<0.05).All above proteins expression has no statistical differences between Flu+ RA group and normal control group(p>0.05).RA had significantly influence on the key proteins of PI3K-Akt pathway in GC-1 cells,while these proteins of PI3K-Akt pathway had no change under the same RA intervention condition.Conclusion: RA could drive spermatogenesis process to normal in cryptorchidism testis when administrated during spermatogenic crucial period.RA may rehabilitate BTB function via RAR?,depressing the PI3K/Akt/m TOR signaling pathway and improving autophagy level in cryptorchidism testis,and recover germ cell function partially.