Endoplasmic Reticulum Stress-induced Autophagy Contribute To Testicular Toxicity Induced By Microcystin-LR

Background Cyanobacteria blooms caused by increased organic nitrogen and phosphorus in water bodies can cause serious pollution of water bodies.Microcystins(MCs)released from cyanobacteria is the most substantial cyanobacterial toxin.Overwhelming evidences show that MC-LR can be accumulated mainly in the liver and induce hepatotoxicity.Recent studies have demonstrated that the gonads are the second most important target organ of MCs after the liver.Previous studies have shown that MCs can induce reproductive toxicity through oxidative stress.However,the mechanism behind interference with gonads caused by MC-LR is still largely unknown.Objective In our experiments,we investigated how MC-LR caused male zebrafishgonad damage and impaired its reproductive capacity,and studied how MC-LR causes endoplasmic reticulum stress and autophagy in male gonad and TM4 sertoli cells.Then,we further studied that MC-LR causes reproductive damage through endoplasmic reticulum stress-induced autophagy.These studies provided a theoretical basis for clarifying the mechanism of MC-LR male reproductive toxicity and providing control measures.Research methods Part 1 MC-LR exposure damages male gonadal and affects male zebrafish reproductive capacity Select 360 3-month-old adult male zebrafish,they were randomly divided into three groups,including the control group,the 5 ?g / L MC-LR group,and the 25 ?g / L MC-LR group.Reared in water containing the corresponding MC-LR concentration.After 30 days of exposure,select 20 of them to mate and spawn.Males were randomly selected to mate and spawn with unexposed females,eggs were collected daily,and calculate their spawning rate,hatching rate,survival rate,deformity rate,heart rate.Dissect the remaining fish and collect their gonadal tissue.Six fish from each group were randomly selected for histomorphological observation of gonad damage.The remaining liquid nitrogen was frozen and transferred to a-80 ° C refrigerator for storage.Part 2 MC-LR induces autophagy in male zebrafish gonadal and mouse testicular support cells(TM4 cells)Use immunohistochemistry to observe zebrafish gonadal damage,LC3 B immunofluorescence staining was used to observe the expression of autophagy-related protein LC3 B,transmission electron microscope was used to observe the changes of subcellular structures such as autophagosomes and endoplasmic reticulum.The expressions of autophagy-related proteins ATG5,ATG12,LC3 B,LC3A,and ULK1 were detected by Western blotting,the expressions of autophagy-related genes ATG5,ATG12,LC3 B,and ULK1 were detected by q RT-PCR.TM4 cell were incubated in the MC-LR at concentrations of 0,7.5,and 30 ?g / ml respectively for 24 h.Then observe the changes of LC3 B expression and lysosomal expression with fluorescent staining.Western blot was used to detect the expressions of proteins related to autophagy,including ATG5,ATG12,LC3 B,LC3A.The expressions of autophagy-related genes ATG5,ATG12,and LC3 B were detected by q RT-PCR.Part 3 Role of endoplasmic reticulum stress-induced autophagy in MC-LR-induced male reproductive injury The well-growth TM4 cells were subdivided into 7 groups: control group,MC-LR group(7.5?g/ml,30?g/ml),autophagy intervention group(3-MA),ERs intervention group(4-PBA),30?g/ml MC-LR+Intervention group(3-MA+30?g/ml MC-LR,4-PBA+30?g/ml MC-LR).Lysosomal immunofluorescence staining was used to observe the changes of the number of lysosomes in the cells.Western blot was used to detect the expression of endoplasmic reticulum stress-related protein GRP78,autophagy-related proteins LC3 A,LC3B and PERK-e IF2?-ATF4 pathway-related proteins p-PERK,PERK,p-e IF2?,e IF2?,ATF4,m TOR,ULK1,ATG5,ATG12.q RT-PCR was used to detect changes in the expressions of endoplasmic reticulum stress-related genes GRP78,autophagy-related genes LC3 B and PERK-e IF2?-ATF4pathway-related genes PERK,ATF4,m TOR,ATG5,and ATG12.Result Part 1 MC-LR exposure damages male gonadal and affects male zebrafish reproductive capacity(1)MC-LR exposure can cause gonadal damage to male zebrafish and affect its fertility:Immunohistochemical observation revealed a slight atrophy of testicular tissue and a slight vacuolation of spermatogenic cells in the 5?g/L group.In the 25?g/L group,thespermatogenic cells in the seminiferous tubules of the testicular tissue were disorderly arranged,and the degree of vacuolization of the tissue became more serious.And as the dose increased,the number of mature sperm also decreased significantly.Transmission electron microscopy observation of zebrafish gonads revealed that autophagosomes were found in the 25 ?g / L group,and the interstitial spaces were enlarged,the tissues appeared hollow,and the mature sperm had abnormal morphology.(2)The spawning test results show that with the increase of the doses,the hatchability of eggs,survival rate of juveniles,and heart rate decreased significantly.Compared with the control group,the malformation rate of juveniles fish in the 5?g/L group was not significantly different,but it increased significantly in the 25?g/L group.Part 2 MC-LR induces autophagy in male zebrafish gonads and TM4 cells.(1)Male zebrafish gonad immunofluorescence showed that LC3 B expression in gonads also increased with increasing dose;The results of TM4 cell immunofluorescence showed similar results,the expression of LC3 B and the amount of lysosomes in the 30?g / ml group increased significantly,autophagy inhibitor 3-MA can inhibit the increase of LC3 B expression and lysosomal volume.(2)Zebrafish gonad protein immunoblotting showed that there was no significant difference in the expressions of autophagy-related proteins ATG5,ULK1,LC3B/LC3 A in the 5?g/L group,but they increased significantly in the 25?g / L group.ATG12 expression was significantly increased in the 5 ?g / L group and the 25 ?g / L group.q RT-PCR detected autophagy-related genes ATG3,ATG5,LC3 B,and ULK1,and found that the expressions of ATG3 and ATG5 increased with increasing doses,and the expressions of LC3 B and ULK1 did not change significantly in the 5 ?g / L group and significantly increased in the 25 ?g / L group.(3)TM4 cell immunoblot experiments show that the expressions of autophagy-related proteins ATG5 and ATG12 increased with increasing dose,compared with the control group,the LC3 B / LC3 A ratio did not change significantly in the 7.5 ?g / ml group,butincreased significantly in the 30 ?g / ml group.q RT-PCR was used to detect the levels of its autophagy-related genes ATG5,ATG12,and LC3 B.The results were similar to the expression of proteins.With the increase of the dose,the expression level increased.After co-treatment with autophagy inhibitor 3-MA,we found that the expression of autophagy-related proteins ATG5,ATG12,LC3 A / LC3 B was inhibited.Part 3 Role of endoplasmic reticulum stress-induced autophagy in MC-LR-induced male reproductive injury The results of western blot and q RT-PCR of TM4 cells showed that the expressions of endoplasmic reticulum stress-related proteins GRP78 increased with the increase of MC-LR exposure dose.The protein expressions related to endoplasmic reticulum stress pathway PERK-e IF2?-ATF4 are also affected.With the increase of MC-LR concentration,the expression levels of p-e IF2? / e IF2? and ATF4 increased.The expression of p-PERK / PERK increased significantly in the 30 ?g / ml group.After using endoplasmic reticulum stress inhibitor 4-PBA to inhibit endoplasmic reticulum stress,compared with the 30 ?g / ml group,the expression levels of p-e IF2? / e IF2?,p-PERK / PERK,ATF4,ULK1,and m TOR all decreased significantly,and the expressions of ULK1 and m TOR increased significantly.The expression level of ATF4 gene was significantly reduced by q RT-PCR.Meanwhile,we also found that the expressions of autophagy-related proteins ATG5,ATG12,LC3 A / LC3 B was inhibited by 4-PBA co-treatment.detection of The gene expressions of ATG5,ATG12 and LC3 B,detected by q RT-PCR,were also suppressed,and the results were consistent with protein expression results.At the same time,immunofluorescence also observed that intracellular MC-LR-induced increase in lysosomal content was also suppressed by4-PBA.Conclusion Exposure of male zebrafish to MC-LR causes damage to its gonads,which in turn willcause damage to reproductive ability,MC-LR can also cause gonadal cell autophagy.Study also found the occurrence of endoplasmic reticulum stress and autophagy in gonadal tissues,in order to explore the relationship between MC-LR induced endoplasmic reticulum stress and autophagy,we further used TM4 cells for experiments.As a result,it was found that inhibition of endoplasmic reticulum stress leads to the inhibition of autophagy and the expression of PERK-e IF2?-ATF4 pathway related proteins and genes.Therefore,it is speculated that MC-LR may induce autophagy in mouse testis sertoli cells through the PERK-e IF2?-ATF4 pathway,thereby causing reproductive damage.