| Objectives: Nowadays,infertility is a common disease affecting human health.About 15% of childbearing couples are plagued by infertility,half of them are caused by male infertility.In male infertility,the treatment of azoospermia is the most intractable.Azoospermia is clinically classified as obstructive(OA)or non-obstructive Azoospermia(NOA).Spermatogenicabilityis severely impaired in NOA patients,which leads to no sperm or only have a bit of sperm.NOA is a disease caused by spermatogenic disorder of testicular,such as testicular capillary dysfunction,testicular germ cell maturation blockage or meiosis stagnation and testicular spermatogenic abnormalities.Although assisted reproduction technology has helped many infertile couples to conceive successfully,many of the NOA patients are still unable to reproduce their genetic offspring.Over the years,the gene mutation or polymorphism related to the NOA has been discovered and become research hotspot.Therefore,an in-depth study of the related genes involved in the regulation of spermatogenesis in NOA patients not only helps to elucidate the etiology of NOA,but also has very important theoretical guidance and application value for the development of target control therapy and assisted reproductive technology for male infertility.CD82 is one of the metastasis suppressors most studied in the field of oncology in recent years.It belongs to the transmernbrance 4 superfamily(TM4SF),with the structure of membrane glycoprotein.It mainly affects the signal transduction between cells and extracellular matrix,and regulates cell proliferation and differentiation.The biological functions of this gene includingregulation of cell cycle,cytoskeleton structure change,protein kinase phosphorylation,cell apoptosis,cell adhesion/migration and so on.Some studies indicated that the expression of CD82 increased in testis of patients combined with NOA.Until now,there is no report about the influence of overexpression of CD82 onabnormalitiesdifferentiation and proliferation of sperm cells,whichprobably lead to sperm formation disorders.In this study,we detected the gene and proteinexpression of CD82 in testis of patients combined with non-obstructive or obstructive azoospermia first,then analysis the difference between the two sets of specimens.Meanwhile,CD82 overexpressed mice model was constructed with injection of adenovirus-associated CD82 vector.Afterwards,we analyzed theeffects of the over-expressed CD82 on the differentiation and proliferation of spermatogenic cells.At last,High Flux Transcription group sequencing method was used to investigate the biological pathways related to spermatogenic disorder cause by CD82 overexpression.This study provides a theoretical basis of further research for the molecular mechanisms related to CD 82 in the differentiation of spermatogenic cells.Methods: 1.HE staining was performed to detect the different morphology of the sperm cells in testicular tissue in azoospermia patients and the expressions of CD82 were tested in testicular tissues in patients combined with obstructive and non-obstructive azoospermia.2.Western blot was conducted to quantified the expressions of CD82 in testicular tissue of obstructive and non-obstructive azoospermia.3.Overexpressed CD82 Adeno-associated virus(AVV)vector was constructed,and testicular tissue of mice were micro-injected with vector to estabalish a model with high-expressed CD82.Meanwhile,trasnsfection efficiency was observed by immunofluorescence.4.HE staining was used to observe the effects of spermatogenesis process in mice testis high-expressed CD82.5.Differentially expressed genes in testis between CD82 over-expressed mice testis and control were identified by high-throughput transcriptome sequencing.6.Six differentially expressed genes were randomly selected,and RT-PCR was performed to verify the resultsof high-throughput transcriptome sequencing.Result: 1.The results of HE staining indicated the morphological changes in the testicular tissue of five azoospermia patients with normal spermatogenic function and seminiferous tubules density,and spermatogenic cells and mature sperm presented in the basal part and lumen of their seminiferous tubular.Other eight patients showed pathological changes with spermatogenic dysfunction and testicular pathology of non-obstructive azoospermatism.The seminiferous tubules were sparse and with thin basal wall,in which that quantity of spermatozoa is significantly reduce,and the number of mature sperm were rarely found.2.The results of Immunohistochemistry staining showed CD82 expressed in interstitial and Sertoli cells in testis tissues of patients combined with obstructive and non-obstructive azoospermatism,and CD82 staining was more remarkable in non-obstructive azoospermatism patients' tissues.Western blot showed that there was a significant difference in CD82 expression between the two groups(P<0.05).3.Recombinant adeno-associated viral CD82 overespressed vector was successfully constructed and then injected into mouse testis tissues.One week later,the results of immunofluorescence showed significant exogenous CD82 expressed in testis of mice in experimental group.4.The results of HE staining of testicular tissues in C57 mice showed the spermatogenic dysfunction in AAV-CD82 injected group,proved by thin lumen walls of seminiferous tubules,and the decreaseof spermatogenic cells.No mature sperm or only a small amount of mature sperm could be seen in the lumen.Oppositely,the control group showed normal spermatozoa and mature sperm in testicular tissues.5.Compared with the control group,the results of transcriptome sequencing results showed thatthere were 590 genes differentially expressed in CD82-overexpressed mice testis,in which 289 genes were upregulated and 301 genes were downregulated.These genes involved in various biological processes,and genesparticipated in cell cycle regulation and transcriptional regulation possess make up a large proportion.Other biological processes involved including cell apoptosis,signaling transduction,immune responses,cell adhesion,and protein phosphorylation etc.6.Six genes were randomly selected from differentially expressed genes pool and then validated by q RT-PCR.The results showed that gene expression levels are consistented with high-throughput transcriptome sequencing.Conclusion: 1.CD82 was positively expressed in the testis of patients with obstructive and non-obstructive azoospermatism.The expression of CD82 in the testis of non-obstructive azoospermatism patients was more significant.2.Overexpression of CD82 in mice testis resulted in spermatogenic dysfunction.Germ cell differentiation is arrested in spermatogonia and primary spermatocytes,suggesting that overexpression of CD82 may cause abnormal spermatogonial differentiation and meiosis dysfunction.3.High-throughput transcriptome sequencing revealed that CD82 overexpression could induce different expression of various genes that may involved in multiple biological processes including cell cycle and transcriptional regulation,which may act as important regulatory roles in CD82-associated dyszoospermia disorder. |
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