Role Of Autophagy In Testicular And Spermatogonial Stem Cell Injury Induced By Hexavalent Chromium In Rats

Hexavalent chromium is a widely-used and harmful poison which has various routes of exposure.In order to observe the damage mechanism of hexavalent chromium on testis and spermatogonial stem cells,a model of hexavalent chromium exposure by combining in vivo and in vitro test was made to explore the role of autophagy in chromium toxicity and further improve the toxic mechanism of chromium poisoning,which will provide a new toxicological perspective for the therapy of chromium poisoning in the clinic.Objective:To study the mechanism of autophagy in testis and spermatogonial stem cell injury induced by hexavalent chromium in rats.Methods:In vivo experiment: 32 male Wistar rats were randomly divided into four groups which were control group and low,medium and high dose potassium dichromate exposure group(0,0.4,0.6 and 0.8 mg/kg of potassium dichromate).Potassium dichromate was injected intraperitoneally to rats for 5 days a week and continuously for 5 weeks.The rats in the control group was given normal saline of equal volume.The behavioral and psychological state of rats in each group were observed.Five weeks later,chromium content in tissues was determined by atomic flame absorption spectrometry,pathohistological changes were observed by HE staining,and the expressions of LC3 B,LAMP1 and LAMP2 proteins were detected by immunohistochemistry,and expressions of autophagy-related proteins MFN-2,PINKI,p-parkin,Beclin1 and LC3 B were detected by Western blotting(WB).Thepositive rate of CHD1 expression,a marker of spermatogonial stem cells,was detected by immunofluorescence chemistry.In vitro experiments: spermatogonial stem cells(SSCs)were isolated and identified as positive for CDH1 and OCT4 expression by cytometry.SSCs were induced to differentiate into sperm.Cytotoxicity of hexavalent chromium was determined by CCK8 assay.SSCs were exposed to 0.5,1.0 and 5.0 mmol/L of potassium dichromate,and SSCs in the control group were treated with the same volume of PBS.24 hours later,the autophagy level in all the groups was determined by electron microscopy,the amount of mitochondria was determined by cytometry,the expression of MFN-2 and LC3 B proteins was detected by double immunofluorescence staining.The expression levels of MFN-2,PINK1,p-parkin,Beclin1 and LC3 B proteins were detected by Western blotting.LDH release from SSCs was measured after 3-MA inhibited autophagy.Results:In vivo experiment: the rats in the control group were in normal condition,while the rats in the exposed group were in poor staus.The index of testis in middle dose group was significantly lower than that of the control group(P < 0.05),and the index of testis in high dose group was significantly lower than that of the control group,the low dose group and the middle dose group(P < 0.05).Atomic absorption results showed that the chromium content in the rats testes of the exposed group was significantly higher than that of the control group,and with the increase of chromium exposure level,there was a significant difference among the groups(P < 0.05).The HE staining results showed that the number of spermatogenic cell layers and the number of mature spermatozoa decreased in the testicular seminiferous tubules of low dose group.In the middle dose group,peritubular membrane thickening,basement membrane fragmentation and extratubular interstitial tissue congestion were also observed.In the high dose group,the spermatogenic structure was completely destroyed,cells were completely absent and multinucleated giant cells were formed.The results of immunohistochemistry and WB showed that the expression levels of PINKI,p-parkin,Beclin1,LC3 B,LAMP1 and LAMP2 protein in testes of chromium exposed group were significantly higher than those of control group(P < 0.05).The expression of MFN-2 protein in the exposed groups was significantly lower than that in the control group(P < 0.05).With the increase of hexavalent chromium exposure dose,the percentage of CDH1-positive cell,a marker of SSCs,became lower and lower(P < 0.05).In vitro experiments: identification results of the cultured cells showed that most of them expressed CDH1 and OCT4 and SSCs were induced to differentiate into sperm.The electron microscopy results showed that the number of autophagic vacuoles in the exposed group was significantly higher than that in the control group(P < 0.05).Mitochondrial quantity in exposed group was significantly lower than that in control group(P < 0.05).Double immunofluorescence staining of MFN-2 and LC3 B showed that the expression of MFN-2 in the exposed group was significantly lower than that in the control group(P < 0.05),whereas the expression of LC3 B in the exposure group was significantly higher than that in the control group.WB results in SSCs groups were consistent with those in vivo.LDH test showed that the cytotoxicity decreased after 3-MA inhibited autophagy of SSCs(P < 0.05).Conclusion:In vivo experiment: hexavalent chromium can cause testicular tissue damage in rats.Within the dose range of this experiment,the level of autophagy gradually increases with the increase of exposure dose and the expression of mitochondrial autophagy-related proteins gradually increases with the increase of exposure dose.In vitro experiments: hexavalent chromium can damage rat spermatogonial stem cells.Within the dose range of this experiment,the autophagy level gradually increases with the increase of exposure dose and the expression of mitochondrial autophagy-related protein increases with the increase of exposure dose.After 3-MA inhibiting autophagy,the toxic effect of hexavalent chromium on spermatogonial stem cells decreased.