| Cadmium(Cd)is a highly toxic environmental heavy metal and endocrine disruptor.Excessive cadmium emission can seriously pollute the environment after it passes through the soil and enters the food chain.The main sources of cadmium exposure in the general population are food and smoking.It has been estimated that the average daily intake of cadmium through human diet ranges from 20.3 to 74.2 μg/day.As a metal ion,cadmium is not easy to be metabolized,and its half-life in the body is as long as 10～33 years.It is classified as a class I carcinogen.Over time,its toxicity induces cell apoptosis through oxidative stress,endoplasmic reticulum stress and calcium homeostasis,which increases the burden of disease in the body,leading to liver and kidney failure,bone injury and reproductive dysfunction.Cadmium can cause male reproductive toxicity.Epidemiological studies showed that semen cadmium concentration was negatively correlated with semen quality and sperm number.In vivo studies showed that cadmium treatment resulted in the destruction of the blood-testes barrier,apoptosis of germ cells,decreased testosterone content,decreased the number of epididymal sperm,increased the malformation rate and reproductive toxicity.In vitro studies showed that cadmium treatment of primary spermatocytes caused the increase of reactive oxygen species(ROS)and induced apoptosis of mitochondria pathway.The main germ cells in testicular tissue are spermatogenic cells,sertoli cells and Leydig cells.Leydig cells participate in the secretion of testosterone,maintain the function of spermatogenic cells,and play a crucial role in male reproductive function.At present,studies on the toxicity of cadmium to the male reproductive system have focused on testicular histology,spermatogenic cells and blood-testis barrier.The data on the cytotoxicity of testicular interstitial cells are limited.It has been reported that cadmium induces apoptosis of Leydig cells through oxidative stress pathway,but the underlying apoptotic mechanism needs to be further explored.Through three quality control mechanisms,including mitochondrial biosynthesis,mitochondrial dynamics(fission and fusion)and mitophagy,mitochondria can change mitochondrial morphology,repair and remove damaged mitochondria,balance ROS production,maintain intracellular mitochondrial homeostasis and regulate cell apoptosis.Previous studies have shown that cadmium exposure affects the apoptosis of liver and kidney cells,nerve cells and primary spermatocytes through mitochondrial hyperdivision or mitophagy pathways.Is the apoptosis process of Leydig cells induced by cadmium a similar pathway?There have been no reports.Based on this theory,this research subjects for cadmium content,select male mice,primary mouse Leydig cells and mouse Leydig cells line TM3 cells as the research object,to explore whether cadmium through the regulation of mitochondrial fusion and the division of the steady state and the way such as mitochondria autophagy induced mesenchymal cells apoptosis,for a comprehensive understanding of male reproductive toxicity mechanism of cadmium to provide new evidence.Part Ⅰ Effects of cadmium exposure on apoptosis and autophagy of Leydig cells in male miceObjective To explore the reproductive toxicity and potential mechanism of mouse Leydig cells exposed to cadmium chloride(CdCl2)in male mice.Methods Twenty 4-week-old male C57BL/6 mice were randomly divided into four groups according to the random value table,with 5 mice in each group.CdCl2 was exposed to different concentrations of 0,0.5,1.0 and 2.0 mg/kg for 28 consecutive days,and the animal model of cadmium exposure was established.Testicular toxicity after cadmium exposure was assessed by HE staining,sperm count,and sperm malformation rate detection.ELISA and immunohistochemistry were used to determine the serum testosterone content and the number of Leydig cells in mice after exposure to cadmium.The apoptosis of Leydig cells in mouse testis tissue sections was analyzed by immunofluorescence combined with TUNEL staining.Changes in autophagosomes and LC3 in Leydig cells of mice were observed using transmission electron microscopy and immunofluorescence.Results The body weight of mice exposed to 2.0 mg/kg cadmium decreased from the second week(P<0.05),while the body weight of mice in the 0.5 and 1.0 mg/kg cadmium treatment groups decreased from the third week(P<0.05).At the end of 4 weeks of exposure,the body weight of mice in all cadmium treatment groups decreased compared with the control group.There was no significant change in the testis/body weight ratio,while the sperm number decreased and the sperm malformation rate increased(P<0.05).Compared with the control group,the serum testosterone content of mice in the cadmium treatment group was decreased,and the structure of spermatogenic tubules was abnormal and spermatogenic cell arrangement disorder.The number of mouse Leydig cells decreased and the apoptosis rate increased with the increase of cadmium exposure dose(P<0.05).Transmission electron microscopy showed,in the cadmium group,that there were more autophagic vesicles,typical double-membrane autophagosomes and less autophagolysosomes in Leydig cells.Immunofluorescence showed that LC3 was expressed in Leydig cells in mouse testis tissue sections,and the expression of LC3 increased with the increase of cadmium exposure.Conclusion Cadmium exposure destroys the structure of testis tissue in mice,reduces sperm count,increases sperm deformity rate,and reduces testosterone content,resulting in reproductive toxicity.Simultaneous cadmium exposure induced apoptosis and regulated autophagy level in mouse Leydig cells.Part Ⅱ Mechanism of cadmium induced apoptosis by excessive mitochondrial fission in mouse Leydig cellsObjective The first part of the study showed that cadmium induced apoptosis of mouse Leydig cells in vivo.This section to TM3 cells(the model cell line of mouse Leydig cells)as the research object.The effect of cadmium exposure on apoptosis of TM3 cells and its possible mechanism were investigate by observing mitochondrial morphological changes,mitochondrial fission and fusion protein expression,mitochondrial function and mitochondria-mediated apoptosis related protein expression.Methods TM3 cells were treated with 5 and 10 μmol/L CdCl2 for 24h(referred to as "cadmium group"),and TM3 cells without CdCl2 treatment were used as control group.CCK-8 assay was used to detect the effect of cadmium on the viability of TM3 cells.The content of testosterone in cell supernatant was determined by ELISA.DCFH-DA probe and MitoSOX were used to detect the ROS levels in cells and mitochondria,respectively.Hoechst33342 staining and flow cytometry were used to observe and analyze cell apoptosis.MitoTracker Red dye was used to detect mitochondrial morphological changes.The localization of cytochrome C(CytC),in cytoplasm and mitochondria was observed by immunofluorescence.JC-1 fluorescent probe detection,ATP detection kit and MitoTracker Green FM dye were used to detect mitochondrial membrane potential,ATP content and mitochondrial quality.Western blotting was used to detect intracellular fission proteins,fusion proteins,CytC and apoptosis related protein.Subsequently,TM3 cells were pretreated with DRP1 inhibitor Mdivi-1(0.1 and 1μmol/L)for 2h,and then treated with 10μmol/L cadmium for 24h.MitoSOX,flow cytometry,Hoechst 33342 staining,MitoTracker dye and Western blot were used to verify the relationship between cadmium-induced TM3 cell apoptosis and mitochondrial excessive division.Results Compared with the control group,the viability of TM3 cells decreased,the testosterone level decreased,the intracellular ROS and mitochondrial ROS levels decreased,and the apoptosis rate increased after cadmium treatment(P<0.05).Mitochondrial morphology became shorter or dotted,and fragmented mitochondria rose(P<0.05).Compared with the control group,the expression levels of mitochondrial fission proteins(DRP1 and FIS1)were significantly increased,while the expression levels of fusion proteins(OPA1 and MFN1)were significantly decreased in the cadmium group(P<0.05).The mitochondrial membrane potential and ATP content in cadmium group decreased with the increase of cadmium dose(P<0.05).Immunofluorescence and Western blot results showed that cadmium treatment increased the level of CtyC protein in cytoplasm and decreased the level of CytC protein in mitochondria(P<0.05),and the level of anti-apoptotic protein Bcl-2 protein in cadmium group was decreased.The expression levels of other apoptosis-related proteins,including cleaved PARP,Caspase-9,cleaved Caspase-9 and cleaved Caspase-3,were significantly increased(P<0.05).Using the DRP1 inhibitor Mdivi-1 to inhibit mitochondrial fission,compared with cadmium group,DRP1 protein expression level was reduced,mitochondrial morphology was partially recovered,mitochondrial ROS accumulation was reduced,apoptosis rate and apoptotic body number were reduced,and the protein level of CytC was increased in mitochondria while that it in was decreased cytoplasm in the inhibitor group.The expression levels of apoptosis related protein cleaved PARP,Caspase-9,cleaved Caspase-9 and cleaved Caspase-3 were significantly decreased(P<0.05).Conclusion Cadmium exposure leads to excessive mitochondrial division in TM3 cells,which then induces the release of CytC and activates the Caspase cascade,leading to cell apoptosis.Part Ⅲ Mechanism of Cadmium inhibition of Mitophagy induced apoptosis in mouse Leydig cellObjective Cells can remove damaged mitochondria by initiating mitophagy mechanism to maintain homeostasis.The first part of this study have been observed in cadmium induced autophagy in leydig cells increased autophagosomes and autophagylysosome was less,while the second part also found when cadmium treatment TM3 cells,the ROS levels rise,mitochondria,divide and lead to damaged mitochondria(shorter form or into dot)increased,speculation may be abnormal mitochondria autophagy functions occur.The damaged mitochondria were not eliminated by mitophagy in time.Is cadmium-induced apoptosis of Leydig cells associated with abnormal mitophagy?In this part,TM3 cell lines,primary mouse Leydig cells and mouse testicular tissues were selected as subjects to explore the changes of mitophagy in Leydig cells after cadmium exposure,the correlation between the changes and apoptosis,and the possible mechanism.Methods TM3 cells and mouse primary Leydig cells were exposed to 0,5 and 10 μmol/L CdCl2 for 24h.The expression levels of mitophagy-related proteins were detected by Western blotting.Apoptosis bodies in TM3 cells were stained with Hoechst33342.Immunofluorescence was used to observe the localization of Parkin in TM3 cells and the localization of NRF1 in mouse testis tissue sections.The cells were pretreated with an autophagy inhibitor rapamycin(RAPA)(50 nmol/L),a lysosomal inhibitor chloroquine(CQ)(10 μmol/L)or a mitophagy inhibitor carbonyl cyanide 3-chlorophenylhydrazone(CCCP)(1Onmol/L)for 2 h and then exposed to CdCl2(10 μmol/L)for 24h.The expression levels of mitophagy-related proteins and related apoptotic proteins were detected by Western blot.The regulatory relationship between NRF1,ATG5 and ATG7 was detected and verified by dual luciferase reporter gene detection,construction of NRF1 protein expression plasmid,protein purification and electrophoretic mobility shift assay(EMSA).Subsequently,the stable transfected cell lines with downregulated expression of Nrf1 expression was contructed,and the mRNA and protein expression levels of ATG5 and ATG7 were measured by RT-qPCR and Western blot.Results Western blotting results showed that the protein levels of autophagy marker proteins LC3-II and p62 were increased in cadmium-treated TM3 cells(P<0.05).Compared with the cadmium group,the protein expression levels of LC3-Ⅱ and p62 in the(Cd+RAPA)group were increased(P<0.05),and the Caspase-3 protein level was significantly increased while the Bcl-2 level was decreased.The protein expression levels of LC3-Ⅱ and p62 in the(Cd+CQ)group were significantly increased(P<0.05),and the number of apoptotic bodies was increased.Western blotting showed that the expression of PINK1 was significantly increased and the protein level of Parkin was decreased in cadmium group(P<0.05).Immunofluorescence showed that mitochondrial colocalization with Parkin was greatly increased in CCCP-treated cells,whereas cadmium decreased mitochondrial colocalization with Parkin in CCCP-treated cells.Immunofluorescence assay showed that NRF1 was highly expressed in Leydig cells,and the expression of NRF1 decreased with the increase of cadmium exposure dose.The results of dual luciferase reporter gene and EMSA assay showed that NRF1 could directly bind to the promoters of Atg5 and Atg7 genes and activate the expression of these two genes(P<0.05).When NRF1 expression was inhibited in TM3 cells,the mRNA and protein levels of Atg5 and Atg7 were significantly decreased(P<0.05).Western blotting showed that the protein levels of NRF1,ATG5 and ATG7 were significantly down-regulated in TM3 and primary mouse Leydig cells exposed to cadmium(P<0.05).Conclusion Cadmium can inhibit NRF1-mediated Atg5/Atg7 expression in Leydig cells,and cause mitochondrial autophagy dysfunction.At the same time,cadmium inhibits the fusion process between autophagosomes and lysosomes,which leads to the arrest of autophagy flux,abnormal process of mitophagy,and apoptosis. |
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