Effect Of Silencing HIF-1? Gene On Testicle Spermatogenesis Function In Varicocele Rats

Objective:Lentivirus vector was constructed to reduce the expression of hypoxia-inducible factor-1?(HIF-1?)gene in testicular tissue of varicocele(VC)rats by CRISPR/Cas9 gene editing technique.The effect of HIF-1?on the function of testicular spermatogenic in VC rats was analyzed by measuring the sperm count and survival rate of epididymis.The morphological structure of testicular tissue was observed by HE staining,and then analyzed the effect of decreasing the expression of HIF-1?on the morphology of testicular tissue in VC rats.The role of HIF-1?in inducing spermatogenic cell apoptosis was detected by detecting apoptosis-related proteins,exploring the mechanism of VC causing abnormal spermatogenic function.Methods:1.Lentivirus vector was constructed and transfected into the testicular tissue of VC ratsThirty male Sprague Dawley rats were divided into the control group(C group),VC model group(V group),VC+HIF-1?-lentivirus group(H group)and VC+Luc-lentivirus group(L group).The VC model was prepared by the classical method of left renal vein partial ligation.Lentivirus vector with low expression of HIF-1?gene was constructed and the titer of the virus was measured and more than 1×10 ~9 TU/ml lentivirus solution was selected to infect rat testis.2.Silence of HIF-1?on testicle of VC ratsThe HIF-1?and luc lentiviruses were injected slowly into the testicles of rats in the H and L groups,respectively.Two months later,the testicular tissue was extracted,and the low knock efficiency of HIF-1?gene at mRNA and protein levels were detected by RT-PCR,western blot and immunofluorescence techniques.3.Changes of testicular spermatogenic function in VC rats before and after the decrease of HIF-1?geneThe left cauda epididymis from rats was cut off after the rats were anesthetized and killed,the spermatozoa were collected by the diffusion method,and placed in a constant-temperature water incubator at 37°C for 10 min.10?l of diluted spermatozoa suspension was dropped onto the counting plate,the sperm concentration and sperm motility were measured by computer-aided sperm analysis.4.Changes of testicular tissue morphology in VC rats before and after the decrease of HIF-1?geneThe left testicular tissue from rats was extracted after the rats were anesthetized and killed.After fixation in Bouin's liquid,dehydration,paraffin embedding,routine dewaxing,and hydration,sections were stained with hematoxylin and eosin at room temperature.The morphology of seminiferous tubules was observed under an optical microscope.5.Changes of spermatogenic cell apoptosis in testicular tissue of VC rats before and after the decrease of HIF-1?geneThe expression of cleaved caspase-3,Bcl-2 and Bax at mRNA level and protein level were detected by RT-PCR,Western blot and immunofluorescence.Results:1.VC model was successfully established,the expression of HIF-1?at mRNA level and protein level in H group were significantly decreased than that in V and L group(p<0.05).No significant difference was observed in HIF-1?expression level between the C group and H group(p>0.05).2.The mean sperm concentration and sperm survival rate in V group were significantly lower than that in the C group(p<0.05).Compared with the V group,the epididymal sperm concentration and sperm survival rate was markedly increased in the H group(p<0.05),no significant difference was observed between the C group and H group(p>0.05).3.Compared with the C group,the seminiferous epithelium was extensively injured,the germ cells were disorderly distributed,and the testicular spermatogenic function was significantly damaged in the V group.The complete degree of seminiferous epithelium in the H group was significantly improved compared with that of the V and L groups,and the structure of seminiferous tubules was complete and the number of spermatogenic cells was increased.4.Pro-apoptotic proteins such as Bax and cleaved caspase-3 revealed higher levels in the V group compared with those in the C group(p<0.05).However,the relative quantity and positivity of Bax and cleaved caspase-3 in the H group were clearly decreased compared to the V group(p<0.05),and no significant difference with C group(p>0.05).By contrast,the expression levels of Bcl-2 in the V group and L group were lower than in the C group and H group(p<0.05).Conclusions:VC causes hypoxia in testis,which further induces the expression of HIF-1?and activates downstream signaling molecules,thereby resulting in increased apoptosis of spermatogenic cells,which affects spermatogenesis and leads to male infertility.By reducing the expression of HIF-1?gene,the downstream signaling pathway as well as the apoptosis pathway of Bcl-2 and caspase-3 may be blocked,we found that sperm count and sperm motility was significantly increased,testicular spermatogenic function was restored and germ cell apoptosis was decreased,it is beneficial to the recovery of testicular function and the improvement of fertility.So,HIF-1?may play an important role in the process of male infertility induced by VC,and the decreased expression of HIF-1?may be considered a predictor of the improvement of hypoxia,which provides a new theoretical basis for the treatment of VC infertility.