Toxicological Effects And Mechanisms Of Rare Earth Yttrium On Male Reproductive System

Objective:China is the largest reserves of rare earths in the world,while the mining and widespread use of rare earth elements（REEs）has led to an increased risk of their potential exposure.Yttrium（Y）,the first heavy rare earth element to be discovered,poses a significant threat to human health.However,studies on the toxic effects of Yon the reproductive system are rare.In this study,we used YCl₃to stain mouse testes and four types of testicular cells（including TM4testicular support cells,TM₃testicular mesenchymal cells,GC-1 mouse spermatogonia and GC-2 mouse spermatocytes）,aiming to clarify the toxic effects of YCl₃in the process of inducing testicular cell damage and toinvestigate themechanism of its toxic effects.Methods:1.YCl₃was administered to C57BL/6 adult male mice for 21 days by acute intraperitoneal injection of the toxin,and the concentration of the toxin was determined by a survival rate experiment.During the modeling period,survival and body weight changes of the mice were observed and recorded daily.Testes were collected immediately after execution,and testicular damage was demonstrated by testicular organ coefficient ratio and H&E staining.The expression levels of markers related to apoptosis,cell scorching,necrosis,and autophagy were detected by Quantitative Real-time PCR（RT-q PCR）,Western-blot and TUNEL staining to clarify the modeof YCl₃-induced testicular death in mice.2.YCl₃was stained with four testicular cells（TM4,TM3,GC-1,GC-2）and the staining concentration was determined using CCK8-half-maximal inhibitory concentrations（IC50）.Testicular cell death was demonstrated using Lactate dehydrogenase（LDH）cytotoxicity assay and cell morphological changes.Annexin V-PI flow cytometry,RT-q PCR and Western-blot assays were used to determine the mode of YCl₃-induced testicular cell death at the cellular level.3.Using TM4 cells as an example,high-throughput gene transcriptome sequencing（RNA-Seq）was performed on YCl₃-stained TM4 cells to clarify that mitochondrial damage and imbalance of Ca²⁺homeostasis may be potential causes of apoptosis and autophagy induced in the cells.4.The mitochondrial membrane potential levels（ΔΨm）of the cells were measured,and the oxidative stress levels,Ca²⁺content and m RNAexpression levels of calcium-related genes were detected in the cells,respectively.The protein expression levels of Nrf2/Ho-1,Camk II/Ampk and NF-κB signaling pathways were detected by Western-blot to clarify the mechanisms of induction of apoptosis and autophagy.5.The reactive oxygen species（ROS）inhibition assay using ROS inhibitor（NAC）pretreatment,Ca²⁺inhibition assay using Ca²⁺chelator（BAPTA-AM）pretreatment,and Ca²⁺back-up assay using NAC and Ca Cl₂co-treatment were used to jointly verify the effects of ROS-Ca²⁺-Camk II/Ampk axis on apoptosis and autophagy from both twosides.6.The relationship between YCl₃-induced apoptosis and autophagy was investigated by using apoptosis inhibitor（Z-VAD-FMK）and autophagy inhibitor（Spautin-1）to inhibit apoptosis and autophagy,respectively,and protein expression of apoptosis and autophagy was detected by Western-blot.Results:1.The body weight of YCl₃-stained mice increased slowly during the injection period and even decreased in 50 and 100 mg/kg YCl₃-stained mice.YCl₃exposure（100 mg/kg）significantly decreased the organ coefficient of the testes.H&E staining showed that the spermatogenic epithelium of the testes of control mice had a normal multilayered structure around the central lumen,which was open and contained numerous spermatozoa.However,the spermatogenic epithelium of the YCl₃-treated mice was detached,obstructed the lumen,prevented sperm formation,with sparse sperm counts and multiple immature sperm cells,and significantly decreased Johnsen scores.YCl₃treatment also contributed to a decrease in m RNA expression of testis-specific genes and blood-testis barrier genes.These results suggest that continuous Yexposure can inducereproductive toxicity and pathological damagein male mice.2.The Bcl-2/Bax m RNAratio was down-regulated in the YCl₃（100 mg/kg）treated group,while the gene expression of the exogenous pro-apoptotic gene Fas and the endogenous pro-apoptotic gene Bcl2l11 was up-regulated.The Bcl-2/Bax protein level ratio was decreased,the c-Caspase3/Caspase3 ratio and the Lc3II/Lc3I ratio were increased.Beclin1 protein expression levels were increased.In contrast,there were no significant changes in Gasdermin-d/c-Gasdermin-d,Caspase1/c-Caspase1,and Mlkl/p-Mlkl,markers of cell scorching and necrosis,respectively.In addition,DNA breaks and degradation in testicular tissues were observed during in situ detection of apoptosis using the TUNEL assay,and the number of positive cells for TUNEL staining was significantly increased.These indicated that YCl₃exposureinduced apoptosis and autophagy inthetestes,butnot cell scorching or necrosis.3.In YCl₃staining experiments with four types of testicular cells,the LDH levels in the400μg/m L YCl₃-treated group were 2-3 times higher than those in the control group,and a significant decrease in the number of cells in the high-dose treated group was also observed microscopically,with massive testicular cell death.The percentage of live cells was significantly reduced,whereas the percentage of early and late apoptotic cells was increased.Western-blot results revealed a dose-dependent increase in the ratio of c-Caspase3/Caspase3and a decrease in the ratio of Bcl-2/Bax.High concentration of Y also increased the expression of autophagy-related proteins（Lc3II and Beclin1）.Similar to the in vivo results,we did not observe the presence of cell scorching and necrosis,as evidenced by the absence of active Caspase 1/Gasdermin-d or MLKL expression.RT-q PCR results showed that the ratio of gene expression of Bcl2/Bax was substantially reduced,and the m RNA expression levels of apoptosis-related Fas,Bcl2l11 and Pmaip1 were significantly increased.Thus,YCl₃induced apoptosis and autophagy in the four types of testicular cells but likewise did not induce cell scorching or necrosis.4.Only TM4 cells in the high concentration group treated with 400μg/m L showed the most pronounced changes in gene expression compared to the control group.Both GO function and KEGG enrichment analysis pointed to Ca²⁺translocation and activation of the Ca²⁺signaling pathway.The heat maps of differentially expressed genes showed that these genes wereclosely associated with mitochondrial RNA（mt RNA）.5.TM4 cells treated with YCl₃showed a significant decrease inΔΨm,damaged mitochondrial membranes,and a significant increase in ROS levels with increasing YCl₃concentration,as well as a significant increase in cellular calcium content.In addition,we found that Nrf2 and its anti-oxidative stress target protein Ho-1 were significantly reduced in YCl₃-treated TM4 cells,while the protein level of Keap1,which acts as an Nrf2 antagonist under physiological conditions,was significantly increased.Simultaneously,we found significantly increased levels of p-Camk II and p-Ampk proteins.However,YCl₃did not induce changes in the NF-κB pathway in TM4 cells,and the expression levels of inflammatory cytokines such as Tnf,Il6,and Il1b genes were not altered in the testes of YCl₃-treated mice.These results suggest that the increase in oxidative stress may be due to the inhibition of the Nrf2/Ho-1 anti-oxidative stress signaling pathway,while the increase in Ca²⁺likely contributes tothe activation of thedownstream Camk II/Ampk pathway.6.NAC（5 m M）partially reversed the YCl₃-induced increase in calcium content,the Camk II/Ampk pathway,apoptosis and autophagy,while significantly inhibiting cellular ROS production.Furthermore,pretreatment with BAPTA-AM（5μM）significantly inhibited calcium and p-Camk II/p-Ampk protein expression levels in TM4 cells while the reduction in intracellular calcium was accompanied by a significant attenuation of 400μg/m LYCl₃-induced apoptosis and autophagy.Correspondingly,Ca Cl₂co-treatment with NAC in the calcium back-compensation assay also partially reversed the decrease in p-Camk II/p-Ampk,apoptosis and autophagy-related protein levels induced by NAC treatment.These results validated that the mechanism by which YCl₃drives apoptosis and autophagy in testicular cells is through activation of the ROS-Ca²⁺-Camk II/Ampk axis.7.Z-VAD-FMK pretreatment inhibited YCl₃-induced apoptosis,but not the development of autophagy.In contrast,Spautin-1 pretreatment resisted YCl₃-induced autophagy,but not apoptosis.These suggest that YCl₃-induced apoptosis and autophagy develop independently of eachother.Conclusion:1.In vivo,YCl₃induced apoptosis and autophagy in mouse testes,which in turn contributed to a decrease in the testis/body mass organ ratio,disrupted the structure of testicular varicocele,hindered sperm formation,and decreased the number of mature spermatozoa and increased the number of immature sperm cells.In vitro,YCl₃exhibited significant toxic effects on all four testicular cell types,as evidenced by significantly increased LDH levels and cell death.As with the in vivo results,YCl₃also induced apoptosis and autophagy in testicular cells,but not cell death forms such as cell scorching and necrosis.2.YCl₃exposure induced significant transcriptional changes in the Ca²⁺pathway and mitochondrial function in testicular cells,which were manifested by decreasedΔΨm,mitochondrial dysfunction,increased oxidative stress（ROS）production,an imbalance in Ca²⁺homeostasis,anda significant increase in Ca²⁺content intesticular cells.3.The increase in ROS was due to inhibition of the Nrf2/Ho-1 anti-oxidative stress signaling pathway,while the increase in Ca²⁺prompted activation of the downstream Camk II/Ampk signal pathway.4.Through ROS inhibition assay,Ca²⁺inhibition assay and Ca²⁺backfill assay,it was verified that increased ROS production prompted by the downregulated Nrf2/Ho-1anti-oxidative stress pathway would in turn induce Ca²⁺homeostatic imbalance and upregulation of the Camk II/Ampk calcium pathway,thus constituting a ROS-Ca²⁺-Camk II/Ampk axis and ultimately inducing cell Apoptosis and autophagy occur,impairing reproductivefunction.5.YCl₃-induced apoptosis and autophagy are not interconnected,and each independently damages the testes and disrupts reproductivefunction.Overall,thestudy suggests that YCl₃exposure intestis leads todisruption of mitochondrial function.In this process,oxidative stress is generated through inhibition of the Nrf2/Ho-1antioxidant stress pathway.The increase in ROS leads to a disruption of calcium homeostasis,possibly by calcium ion efflux from the endoplasmic reticulum or influx from the extracellular compartment,ultimately leading to a substantial increase in calcium levels within the mitochondria.Finally,the increase in intracellular Ca²⁺leads to the independent development of apoptosis and autophagy through activation of the downstream Camk II/Ampk signaling pathway,which ultimately damages the testis and affects reproductive function.