The Role Of ATG 12-mediated Sertoli Cell Autophagy In 5-HMF Induced Blood-testis Barrier Disruption And Its Underlying Mechanisms

5-hydroxymethyl-2-furfural(5-HMF),a toxic compound isolated and purified from cigarette smoke extract(CSE)of Kentucky reference cigarettes 3R4 F,was found to be metabolically activated by Cytochrome P450 2A13(CYP2A13)in our previous study.5-HMF was the dominant component of tobacco as its total amount identified in CSE was 117.5 μg/cig,which was comparable with nicotine(103.7 μg/cig)and approximately 10,000 times higher than the content of NNK.And moreover,the metabolic activity of CYP2A13 for 5-HMF was similar to that for NNK,suggesting that 5-HMF might be a significant toxic component in cigarette smoke.Furthermote,in our previous experimental research respecting the deleterious effect of 5-HMF on respiratory system of mice,the disrupted seminiferous tubule,reduced epididymal spermatozoa and decreased sperm motility were surprisingly observed.Smoking-induced deterioration of male semen has been well accepted for the public,but due to the complexity of cigarette smoke components,the existing research on the association between male reproductive toxicity and key component of cigarette smoke was extremely limited.Therefore,we preliminarily speculated 5-HMF as a valuable and important component which could exert key role in cigarette smoke induced male reproductive damages.The blood-testis barrier(BTB)is the first parclose that protect the testis from permeating and damaging of exogenous toxic substances.Sertoli cell plays key rolein the formation and function of the BTB.And the circulatory toxic substances will enter into the seminiferous duct and directly destroy the spermatogenic cells once sertoli is damaged.Our pervious study also observed that sertoli cell was a potential target for environmental pollutants(such as PFOS)induced reproductive dysfunctions.Thus,in this study,we mainly recongnized sertoli cells as research targets and focused on the deleterious of 5-HMF on sertoli cells and BTB.Considering the key impact that autophagy may exert in physiological and pathological processes of cell damages and the popular tissue that autophagic research stand in the current life science,we therefore explored the association between autophagy and 5-HMF induced sertoli cell dysfunction and BTB to further verify whether 5-HMF can be considered as an important component of cigarette smoke which can damage male reproduction.In this study,a 5-HMF nasal exposure model was conducted by using a small animal aerosol inhalation exposure device.By evaluating the general reproductive damage,reproductive hormone levels and abnormal structure and function of BTB in 5-HMF treated mice,the pattern of 5-HMF-induced male reproductive damage and the possibility of Sertoli cells/BTB as potential 5-HMF targets were identified.Moreover,the effects of 5-HMF on the production and accumulation of autophagosomes in testis and Sertoli cells,the changes of autophagic flux and autophagic pathways,especially ATG12-mediated pathways were analyzed by using the mice and primary sertoli cells treated with 5-HMF.On this basis,the whole-body atg12 knockout(atg12+/-)mice was conducted to explore the influence of decreased ATG12 expression on the testicular toxicity of 5-HMF and the key role of ATG12-mediated autophagy in 5-HMF induced BTB dysfunction.Part Ⅰ: Effects of 5-HMF on spermatogenesis and BTB functions in mice Obiective: To clarify the pattern of 5-HMF-induced male reproductive damage,and to evaluate the possibility of Sertoli cells and BTB as potential targets upon 5-HMF exposing.Methods: Adult(10-12 weeks)male C57BL/6 mice were intranasally administrated with aerosol 5-HMF for 60 consecutive days.The body weight and gonadal organ coefficients were detected to evaluate the general toxicity.The hematoxylin-eosin(HE)stain was applied to explore the testicular and epididymal morphology and the computer-assisted sperm analysis(CASA)was used to evaluate the sperm parameters.The ELISA was applied to analyze the changes of hypothalamic-pituitary-testicular(HPT)axis.In addition,transmission electron microscopy(TEM)and biotin tracer analysis were performed to decte the structure and function of BTB.And immunoblotting was used to evaluate the expression of BTB related proteins.Results: 5-HMF exerted no influence on bodyweight and gonadal organ(testis and epididymis)coefficients but could result in histological abnormity like thin seminiferous epithelium and vacuolization of spermatogenic cells.5-HMF significantly decreased sperm counts and motility without influencing the levels of testosterone(T),estradiol(E2),follicle-stimulating hormone(FSH)and luteinizing hormone(LH).Moreover,the sertoli cell tight junctions(TJs)and BTB might be potential targets as 5-HMF could induce BTB structure disruption like shallow shading,fracturing and disintegrating and could also induce BTB dysfunction by allowing the more permeation of biotin tracer.The expressions of TJ proteins ZO-1,Occludin and Claudin-11 were significant decreased to 85%,48% and 57% respectively while no downregulation was observed in expressions of Cox-43,β-catenin and α-catenin.Part Ⅱ: Effects of 5-HMF on testicular and sertoli cellular autophagy Obiective: To investigate the effects of 5-HMF on autophagosomes,autophagic pathways and autophagic flux in testis and Seroli cells,and to systematically explore the molecular mechanisms by which 5-HMF induce autophagic pathways.Methods: Based on the in vivo and in vitro primary sertoli cells 5-HMF exposing model,the production and accumulation of autophagosomes were observed by TEM analysis.The expressions of autophagic genes and proteins were detected by q RT-PCR and immunoblotting.In addition,the mouse sertoli cell line TM4 was transfected with m RFP-GFP-LC3 and exposed to 5-HMF to determine the autophagic flux.Combined treatment of 5-HMF and CQ was further used to verify autophagic flux by immunoblotting and Immunofluorescence analysis.Results: 5-HMF could induce the production and accumulation of monolayer autolysosomes and bilayer autophagosomes with cytoplasm,proteins and organelles in sertoli cells.Moreover,5-HMF sifnificantly decreased the expression of mtor and increased the expressions of atg5,atg12,atg3,atg10,atg7,and among them atg12 was the most affected gene with a 9 times higher than controls.Further protein analysis confirmed the up-regulation of ATG12 and the subsequent LC3 II/I autophagy.Increased autophagic flux was observed in 5-HMF treated m RFP-GFP-LC3-TM4 cells with an increasing number of autolysosomes,autophagosomes and down-regulation of autophagic substrate P62.CQ could also aggravated LC3 II/I up-regulation induced by 5-HMF further indicated the enhancement of autophagic fulx.Part Ⅲ: Effects of ATG12-mediated autophagy on 5-HMF induced BTB disrutpion Obiective: To explore the role of atg12+/-on 5-HMF-induced spermatogenesis destruction and BTB disrutpion,and to elucidate the relationship between ATG12-mediated Sertoli cell autophagy and the degradation of TJ proteins induced by 5-HMF.Methods: atg12+/-mice was obtained by using CRISPR/CAS9,and immunoblotting was applied to validate the down-regulation of ATG12 in major organs.Based on the previous exposing condition,WT and atg12+/-mice were treated with 5-HMF to detect the changes of increased autophagosomes and autophagic proteins by TEM and immunoblotting of autophagic proteins;to explore the alterations of disturbed spermatogenesis by light microscopy and CASA analysis;and to analyze the influence of haploinsufficiency of ATG12 on structure and function of BTB by TEM,biotin tracer and immunoblotting of TJ prtoteins.In addition,primary sertoli cells isolated from WT and atg12+/-mice were treated with 5-HMF and/or CQ to determe the transepithelial electrical resistance(TER)and TJ proteins expression.Finally,TM4 was treated with 3-MA and/or 5-HMF to detect the influence of autophagy inhibition on expression and location of TJ protein by immunofluorescence.Results: Compared with the WT control mice: 1.Knockout confirmation: atg12+/-sigfnificantly decreased ATG12 expression in testis,epididymis,liver and lung,of note,the testis expression decreased by 50%.2.Autophagic evaluation: 5-HMF-induced increment in autophagosomes,ATG12 and LC3 II/I expressions was partially abolished by haploinsufficiency of ATG12.3.General testicular toxicity: 5-HMF could induce thin seminiferous epithelium,decreased sperm counts and motility,which could be attenuated by haploinsufficiency of ATG12.4.Function and structure of BTB: 5-HMF significantly disrtupted TJ structures by shallow shading,fracturing and disintegrating;destroried BTB functions by allowing the more permeation of biotin tracers;and down-regulated TJs expressions,which were reversed by haploinsufficiency of ATG12 Futhermore,as for primary sertoli cells,5-HMF could also decrease the TER and down-regulate TJ proteins and haploinsufficiency of ATG12 could partially rescue the injury.And 5-HMF was found to transfer Occludin into the cytoplasm and reduce its fluorescence intensity in TM4 while combined treatment with 3-MA could abolish the decreasing of fluorescence intensity without effect on Occludin endocytosis.Conclusion To explore the role of ATG12-mediated autophagy in 5-HMF induced BTB disruption and its molecular mechanism,the structures and founctions of sertoli cells and BTB in 5-HMF treated mice were firstly evaluated.The autophagic flux and pathway analyses were further performed to determine the role of ATG12 and its related autophagy.And finally,the testicular phenotype differences between 5-HMF treated WT 和 atg12+/-mice were systematically explored.The major findings were listed as follows: 1.Aerosol inhalation of 5-HMF could directly destroy the testis of mice,inducing disruption in spermatogenic epithelium,decrease in sperm counts and motor ability without changing the levels reproductive hormones.It was suggested that 5-HMF might be an important reproductive toxic component from cigarette smoke,and its effect may be direct damage to testis rather than endocrine regulation.2.Sertoli cells and BTB might be the direct targets of 5-HMF,as it could destroy the tight junction by interfering with the classic "sandwich" structure and "anti-permeation" function of BTB.5-HMF mainly caused down-regulation of TJ proteins like ZO-1,Occludin and Claudin-11 in sertoli cells,suggesting that tight junction may be the main target upon 5-HMF exposing.3.5-HMF could induce the accumulation of autophagosomes and the increment in testicular and sertoli cellular autophagic flux.Up-regulation of ATG12 might play a key role in 5-HMF-induced sertoli cell autophagy.4.Haploinsufficiency of ATG12 could partially ameliorate the spermatogenesis disruption,BTB dysfunction,TER reduction and TJ proteins down-regulation caused by 5-HMF.Autophagy pathway inhibitors CQ and 3-MA could also alleviate the BTB/TJ damage induced by 5-HMF,suggesting that ATG12 and the subsequent autophagy activation of Sertoli cells could be a key molecular pathway in degradation of TJ proteins induced by 5-HMF.