Roles And Molecular Mechanisms Of DNA Damage Repair Gene Xrccl And Selected Nanometer Materials On Male Reproductive System

Infertility has always been a major scientific issue in the reproductive health research field.According to WHO prediction,infertility will be the third-largest disease next to cancer and cardiovascular disease in the 21st century.Reportedly,for half a century,human sperm quality significant decreased,spermatogenic failure had become the most common etiology.And it is the main threat to male reproductive health in our country.Spermatogenic failure may be closely related to genetic modification and environmental chemicals exposure.Spermatogenesis is a continuous cell division and differentiation process,it includes spermatogonium proliferation,spermatocyte meiosis,and spermatid metamorphosis,and it forms mature sperm at last.During the complicated process,DNA damage repair mechanism is an important self-repair ability to reply endogenous and exogenous damage.In our labouratory,we found DNA repair gene X-ray repair cross-complementing gene 1(XRCC1)polymorphisms contributed to the risk of developing idiopathic azoospermia in a selected Chinese population.However,the molecular mechanism of it remains unclear.Therefore,we structured Xrccl knock out mice model to explore the role of Xrccl in the spermatogenesis.Meanwhile,due to the stable human genetic material,the gene contributed to spermatogenesis failure risk was limited,thus environmental chemicals may impaired the spermatogenesis.Therefore we selected nanometer materials to evaluate its male reproduction effect.Our research hypotheses were:a)DNA damage repire gene Xrccl has crucial roles in the spermatogenesis in mice;b)selected nanometer materials may affect DNA in germ cells and then damage spermatogenesis.To demonstrate above hypothesis,firstly we structured conditional Xrccl knock out in the primordial germ cell to investigate the male reproductive function.Then,research the mechanism of Xrccl in the spermatogenesis with mice spermatogonia stem cell model.Finally study the role of environmental factor in the spermatogenetic failure by evaluating the effect of nanometer materials in the germ cells in vitro.PartⅠRoles and Molecular Mechanisms of DNA Damage Repair Gene Xrccl on Male Mice Reproductive FunctionObjectiveXRCC1,has no known enzymatic activity,interacts with many proteins known to be involved in DNA damage repair process.Previous researches have proved that the presence of DNA damage repair is important in germ cells.However,the exact role of Xrcc1 during the spermatogenesis and maintenance of the reproductive system remains elusive.MethodsWe have generated a mouse inactivation of the orthologous murine gene(Xrccl)by Cre-Loxp technique in primordial germ cell to enhance our understanding of the roles and mechanisms ofXrcc1 in the spermatogenesis process with reproductive function evaluation.ResultsThe homozygous null male mice presented completely infertility,as evidenced by mating experiments and sperm parameters.We found profound pathological alterations in seminiferous tubule section.Loss of Xrccl also led to persistence of apoptosis,mitochondrial dysfunction and increased oxidative stress throughout the testis tissue.Additionally,we observed the the disruption of germ cell differentiation in the absence of Xrccl.ConclusionThis study provided the first line of evidence that Xrccl could maintain spermatogonium sternness,mitochondrial function and reduced apoptosis in mice testes,suggested it participating in spermatogenesis process.Part ⅡRoles and Molecular Mechanisms of DNA Damage Repair Gene Xrccl in the Mice Spermatogonial Stem CellsObjectiveBased on our above in vivo results,we speculated loss of Xrccl result in mitochondrial dysfunction via oxidative stress mechanism.Thus we investigated the roles of Xrccl in the mouse spermatogonial stem cells(SSCs).MethodsFirstly we built mouse SSCs model and introduced Xrcc1 siRNA into an adenovirus.We explored the mitochondrial parameters on the SSCs with and without Xrccl knock down and N-acetyl-L-cysteine(NAC)administration.ResultsXrccl deficient increased oxidative stress status and impaired mitochondria function in SSCs.NAC supplementation could partial rescue cellular oxidative stress and mitochondria function.Although loss of Xrccl reduced SSCs sternness markers and spermatogenesis related transcripts,the molecular mechanism remianed unknow.ConclusionXrccl deficient in SSCs could impared mitochondrial dysfunction via oxidative stress pathway and decreased stemness and spermatogenesis by unknown ways.Part ⅢEffects of Selected Nanometer Materials on DNA of GC-2spd CellsChapter I Effects of Multi-walled Carbon Nanotubes on DNA of GC-2spd CellsObjectiveMulti-walled carbon nanotubes(MWCNTs)have been widely used in many fields and were reported to cause reversible testis damage in mice at high-dose.However the reproductive effects of low dose MWCNTs remained elusive.MethodsHerein,we used the mice spermatocyte cell line(GC-2spd)to assess the reproductive effects of MWCNTs.Size distribution,zeta potential,and intensity of MWCNTs were characterized.ResultsA maximal concentration of 0.5 μg/mL MWCNTs was found to be nonlethal to GC-2spd.At this dose,cell cycles and the ROS levels were in normal status.We also found MWCNTs accumulated in mitochondria,which caused potential mitochondrial DNA damage in spermatocyte.Furthermore,the expression level of mitochondria-related genes,the oxygen consumption rate,and cellular ATP content were declined compared to controls,even at the nonlethal dose.ConclusionOur results suggested for the first time that,in germ cells,mitochondrion was a cellular organelle that accumulated MWCNTs.Chapter II Effects of Zinc Oxide Nanoparticles on TM-4 Cells and DNA of GC-2spd CellsObjectiveZinc oxide nanoparticles(ZnO NPs)were widely application in the field of life in recent years.Most investigators focused on the effects of ZnO NPs on human health,while limited information was available on the male reproductive system.MethodsHerein,mice Sertoli cell line(TM-4)and mice spermatocyte cell line(GC-2spd)were used as in vitro models to explore the reproductive effects of ZnO NPs at sublethal dose and its underlying mechanisms.ResultsCells were treated with different concentrations of ZnO NPs.By cell viability assay,a dose of 8 μg/mL was found as a sublethal dose and increased the ROS levels in both cells.The decreased glutathione level and increased MDA level were also found in ZnO NPs treated group.In TM4 cells,the expressions of BTB proteins(ZO-1,occludin,claudin-5,and connexin-43)were lower in the ZnO NPs group.The increased cell permeability and increased TNF-a secretion were also observed in ZnO NPs group.In GC-2spd cells,S phase arrest and DNA damage occurred in ZnO NPs group,which could be partially rescued by NAC.ConclusionOur findings demonstrated that exposure to ZnO NPs induced ROS generation,caused DNA damage of germcells,and down-regulated the expression of BTB proteins in Sertoli cells which could compromise the integrity of the blood-testis barrier.All these contributed to the male reproductive cytotoxic effects of ZnO NPs that could be partially rescued by anti-oxidants.