Study On The Mechanisms Of Defective Spermatogenesis Induced By TiO₂ NPs

Objective:Titanium dioxide nanoparticles（TiO₂ NPs）exposure can cause defective spermatogenesis and reduce sperm count.However,due to the complexity of spermatogenesis,the mechanisms of the defective spermatogenesis induced by TiO₂ NPs are still unclear.The 3D blood-testis microfluidic chip was established and combined with transcriptomics and metabonomics to better study the molecular mechanisms of TiO₂ NPs-induced defective spermatogenesis.Methods:3D blood-testis microfluidic chip is constructed by soft lithography technology.The expression of ZO-1 in the Sertoli cell（TM4）unit was detected with immunofluorescence to evaluate the integrity of the barrier structure of the chip.The enzyme-linked immunosorbent assay was used to analyze the glucose content in the media supernatants of the chip outlet to evaluate the system stability of the chip.After the chip was treated with 100μg/m L TiO₂ NPs for 24 h,TM4 and spermatogonia cells（GC-1）were respectively collected for transcriptomics and metabonomics.And using“Metabianalyst”to find the biological processes,key signal pathways,key genes,and key metabolites related to spermatogenesis in the two cells.Then western blot and commercial kits were used to verify the changes in key protein expression levels and metabolite content.Result:1.The biomimetic 3D blood-testis microfluidic chip was successfully constructed,which includes TM4,GC-1,and endothelial cell culture units.The expression level of ZO-1 protein in TM4 cells was detected in the Sertoli cell unit,and the fluorescence intensity of ZO-1 protein was strong.In addition,the glucose concentration in the media supernatants of the chip outlet was monitored,and found that the glucose concentration of the 3D chip remained stable at 24-72 h.2.After the 3D blood-testis microfluidic chip was treated with TiO₂ NPs,the GO enrichment of transcriptomics analysis in TM4 cells showed that DEGs were mainly related to the monosaccharide metabolic process,glucose catabolic process,and hexose catabolic process.The KEGG enrichment of these DEGs was mainly involved in the HIF-1 signaling pathway and glycolysis/gluconeogenesis.Moreover,the KEGG enrichment results of the metabonomics analysis showed that the differential metabolites were mainly involved in fructose and mannose metabolism and the glucagon signaling pathway.The joint pathway analysis of DEGs and differential metabolites showed that TiO₂ NPs exposure affected glycolysis/gluconeogenesis of TM4 cells.The main source of energy substrate in spermatogenesis is verified that produced by TM4 cells under the action of lactate dehydrogenase A（LDHA）through glycolysis.Therefore,the key genes and metabolites of the verified production process were further searched,and it was found that the expression levels of HK1,PFKM,and LDHA,and the content of fructose 1,6-2 phosphate in the TiO₂ NPs group were significantly reduced compared with the control group（P<0.01）.The protein expression levels of HK1,PFKM,and LDHA,and the content of fructose 1,6-2 phosphate and lactate were significantly reduced too,which were validated with western blot and commercial kits.3.After the 3D blood-testis microfluidic chip was treated with TiO₂ NPs,the GO enrichment results of transcriptomics analysis in GC-1cells showed that DEGs were mainly related to the processes of behavioral fear response and behavioral defense response.The KEGG enrichment of DEGs was mainly involved in the chemokine signaling pathway.Moreover,the KEGG enrichment results of the metabonomics analysis showed that the differential metabolites were mainly involved in oxidative phosphorylation.The joint pathway analysis of DEGs and differential metabolites showed that TiO₂ NPs affected the chemokine signaling pathway and glutathione metabolism related to cell proliferation of GC-1cells.The verified results of the key proteins and metabolites showed that the protein expression levels of Cxcl 13,Stat 3,and p-Stat 3 and the cell proliferation rate were significantly decreased（P<0.05）,and the expression level of GSR and GPX4 proteins and the content of GSH were significantly increased in the TiO₂ NPs group were significantly decreased compared with the control groups（P<0.05）,which were consistent with the multi-omics analysis.Conclusion:TiO₂ NPs exposure can reduce the energy substrate supply required for spermatogenesis by inhibiting the production of lactate of TM4 cells.In addition,TiO₂ NPs also inhibit the proliferation of GC-1 cells through suppressing the chemokine signaling pathway to decrease the expression levels of p-Stat before the occurrence of oxidative damage.Inadequate supply of energy substrate in spermatogenesis and slow division of spermatogonia induced by TiO₂ NPs are the fundamental reasons for TiO₂ NPs-induced defective spermatogenesis.