Molecular Mechanism Of Mitochondrial Fission In Male Reproductive Toxicity Induced By Nickel Nanoparticles

Male reproductive disorders and declining fertility rates play an important role in birth rates,and their impact on future populations makes them important public health issues this century.Defects in spermatogenesis are the most common manifestation of male infertility,and exposure to environmental pollutants has been suggested as a potential cause.Nickel nanoparticles(Ni NPs),which are metallic nanomaterials that combine the properties of nickel and many other new properties,are widely used in various fields.However,its potential harm cannot be ignored.Our research group has been working on the toxicity of Ni NPs for a long time.Previous studies have found that Ni NPs can stimulate oxidative stress in rats and mice,inducing germ cell apoptosis and abnormal spermatogenesis and causing mitochondrial damage,during which the mitochondrial apoptosis pathway and PI3K-AKT signaling pathway are involved in the regulation of Ni NPs-induced apoptosis.Mitochondria maintain size and mass through fission and fusion,which are essential for cell survival.For this reason,we speculate that mitochondria are the targeted organelles in the toxic effects of Ni NPs.Thus,this study aimed to explore the male reproductive toxicity of Ni NPs and the role of mitochondrial fission in mouse spermatogonia(GC-1 spg)and mouse spermatocytes(GC-2 spd(ts)).Furthermore,the regulatory effect was reversed using Mdivi-1 and lentiviral infection,respectively.1.Study on the toxicity of Ni NPs on reproductive cellsScanning electron microscope(SEM)and transmission electron microscope(TEM)demonstrated that Ni NPs were spherical particles with diameters between 25～125 nm,and the distribution was not uniform.The results of nanoparticle particle size analyzer showed that the Zeta potential of 25 μg/mL Ni NPs was-4.50 ± 0.26 mV,and the hydrated particle size distribution range was 220.2～531.2 nm with a peak value of 391.1 nm;the Zeta potential of 50μg/mL Ni NPs was-6.01 ± 0.33 mV,and the hydrated particle size distribution ranged from255.0 to 712.4 nm with a peak value of 408.7 nm;the Zeta potential of 100 μg/mL Ni NPs was-7.60 ± 0.43 mV,and the hydrated particle size distribution ranged from 220.2 to 955.4 nm with a peak value of 562.1 nm.All these data show that the Ni NPs suspension has poor stability and agglomeration phenomenon.GC-1 spg cells and GC-2 spd(ts)cells were used as the research objects,and 0μg/mL(control group),25 μg/mL,50 μg/mL,and 100 μg/mL Ni NPs groups were set.Cell morphology,intracellular ultrastructure and mitochondrial morphology were observed.Cell viability and apoptosis were detected.Intracellular adenosine triphosphate(ATP)content,reactive oxygen species(ROS)content,mitochondrial reactive oxygen species(MtROS)content,and mitochondrial membrane potential(MMP)levels were determined.Simultaneously,the expression levels of mitochondrial fission/fusion-and apoptosis-related proteins were determined.CCK-8 assay and flow cytometric apoptosis assay showed that Ni NPs could dose-dependently inhibit the viability of GC-1 spg cells and GC-2 spd(ts)cells,and increase the overall apoptotic rate of both cells(P < 0.05).The observation of cell morphology showed that with the increasing Ni NPs concentration,the morphology of GC-1 spg cells and GC-2 spd(ts)cells gradually became rounded and broken,the cell growth density decreased and the intercellular junctions were not tight.The observation of intracellular ultrastructure and mitochondrial morphology showed that the mitochondrial morphology of GC-1 spg and GC-2spd(ts)cells in the Ni NPs group was fragmented,and the mitochondrial cristae structure was broken or reduced.In addition,compared with the control group,ROS and MtROS accumulated in GC-1 spg and GC-2 spd(ts)cells in the Ni NPs group,and the levels of ATP and MMP decreased(P < 0.05),indicating that Ni NPs can cause reproductive intracellular mitochondrial dysfunction.Western blot assay showed that compared with the control group,the relative expression levels of Drp1,Fis1,Bax,Caspase-9,and Caspase-3 proteins increased significantly in GC-1 spg and GC-2 spd(ts)cells(P < 0.05),the relative expressions of proteins Mfn1,Mfn2,Opa1 and Bcl-2 remarkably decreased(P < 0.05),and the ratio of Bax/Bcl-2 was significantly higher in the 25,50,and 100 μg/mL Ni NPs groups(P < 0.05).All these data suggest that Ni NPs promote germ cell apoptosis,which may be associated with enhanced mitochondrial fission.2.Regulation of mitochondrial fission by Mdivi-1 on the reproductive toxicity of Ni NPsUsing GC-1 spg and GC-2 spd(ts)cell models,cell viability was separately detected by CCK-8 assay to confirm the optimal concentration of Mdivi-1 after treatment with different concentration of Mdivi-1.Firstly,control group,Mdivi-1 group,Ni NPs group and Ni NPs+Mdivi-1 group were set.Then cell apoptosis in GC-1 spg cells and GC-2 spd(ts)cells,the changes of cell mitochondrial morphology,the levels of intracellular ROS,MtROS,ATP and MMP,as well as the expressions of intracellular apoptosis and division-related proteins were carried out to explore the important mechanism underlying the mitochondrial fission in the process of Ni NPs induced-apoptosis.The results showed that the optimal concentration of Mdivi-1 was 15 μM in both GC-1 spg cells and GC-2 spd(ts)cells.Compared with the Ni NPs group,the apoptosis rate was significantly reduced(P < 0.05),the state of broken mitochondria was significantly improved,the accumulated ROS and MtROS were significantly eliminated(P< 0.05);intracellular ATP and MMP levels were significantly increased(P < 0.05);intracellular over expressed proteins Drp1,Fis1,Bax,Caspase-9,and Caspase-3 were inhibited,and the under-expressed protein Bcl-2 was improved,with a significantly decreased ratio of Bax/Bcl-2in the cells of Ni NPs+Mdivi-1 group(P < 0.05).All these results indicate that Mdivi-1 can effectively inhibit Ni NPs-induced enhancement of mitochondrial fission in germ cells and reduce the occurrence of apoptosis.3.Effects of low expression of Dnm1 l on the reproductive toxicity of Ni NPs by regulating mitochondrial fissionUsing GC-1 spg and GC-2 spd(ts)cell models,the optimal conditions were explored for lentivirus-transfected cells with the low expressing Dnm1 l by the bio-company and to construct a cell model with low expression of Drp1(Dnm1l-KD)and the corresponding negative control(Dnm1l-NC).Thereafter,the control group,Ni NPs group,Ni NPs+Dnm1l-NC group and Ni NPs+Dnm1l-KD group were set.The results showed that the optimal infection conditions were the equivalent of 50 multiplicity of infection(MOI)for lentivirus infection of GC-1 spg cells,supplemented with Hitrans G A infection enhancer,and the equivalent of 20 MOI for the low expression of Dnm1 l lentivirus infection of GC-2 spd(ts).Compared with 100 μg/mL Ni NPs alone,the combination of Ni NPs with Dnm1l-KD significantly decreased the apoptosis rate of GC-1 spg and GC-2 spd(ts)cells(P < 0.05),improved intracellular damaged mitochondrial morphology,reduced the intracellular ROS and MtROS contents(P < 0.05),increased the intracellular ATP and MMP levels(P < 0.05),downregulated the expression levels of intracellular Drp1,Fis1,Bax,Caspase-9 and Caspase-3 proteins,and upregulated the expression level of protein Bcl-2,thereby decreasing the ratio of Bax/Bcl-2(P < 0.05).All these results show that low expression of Dnm1 l inhibits apoptosis and alleviates cytotoxic effects through inhibiting Ni NPs-induced mitochondrial fission.Taken together,Ni NPs exposure causes morphological and structural damage and dysfunction of targeted organelles(mitochondria)in GC-1 spg and GC-2 spd(ts)cells and disrupts intramitochondrial homeostasis,thereby resulting in enhanced Drp1-mediated mitochondrial fission and cell apoptosis,along with aggravated cytotoxicity and obvious reproductive toxicity.The inhibitor Mdivi-1 and lentiviral-transfected low expression of Dnm1 l can significantly alleviate the germ cell toxicity induced by Ni NPs,suggesting a certain therapeutic effect.Therefore,this study offers a reference for expanding the reproductive toxicity effect of Ni NPs and potential molecular mechanism and provides an important basis for finding potential targets and treatment of Ni NPs.