The Role Of Endoplasmic Reticulum Stress And Its Mediated Endoplasmic Reticulum Autophagy In Cadmium-induced Testosterone Synthesis Disorder In Leydig Cells

Cadmium(Cd)is a widespread toxic heavy metal in nature.Testis is one of the main target organs of cadmium toxicity due to its high sensitivity to cadmium in male reproductive system.The male reproductive toxicity of cadmium is mainly aimed at two types of specific cells in the testis,namely,Sertoli cells and Leydig cells.Leydig cells mainly promote spermatogenesis and maintain the normal growth and development of males by secreting testosterone.Previous studies of the research group have shown that cadmium exposure can lead to testosterone synthesis disorders in male animals.Endoplasmic reticulum(ER)is the main site of testosterone synthesis in Leydig cells.In order to further explore the molecular mechanism of cadmium-induced testosterone synthesis disorder in male animals,on the basis of constructing cadmium poisoning models in vivo and in vitro,this experiment studied the role of endoplasmic reticulum stress(ERS)and its mediated autophagy of endoplasmic reticulum(ER-phagy)in cadmium induced testosterone synthesis disorder in male animal leydig cells.1.Effects of cadmium on testicular and testosterone secretion in ratsIn order to explore the effects of cadmium exposure on the testis and epididymis of male animals,12 SD male rats aged 6 weeks were selected for the experiment and divided into groups and treated as follows:control group(Con),cadmium exposure group(Cd),6 rats in each group.Mice in the cadmium exposure group were exposed to 75mg/L cadmium water for free drinking water to establish a 4-week model of poisoning in vivo.The content of cadmium in testicular tissue was detected by flame atomic absorption spectrometry.The organ coefficients of testis and epididymis were calculate.The sperm motility,motility and movement parameters were analyzed by comprehensive semen analysis system.Pathological changes of testis and epididymis were observed by HE staining.The testicular spermatogenic ability was analyzed by Johnsen score.The testosterone secretion level in serum was detected by ELISA;testosterone synthesis related protein expression was detected by Western Blot.The ultrastructural changes of Leydig cells were observed by transmission electron microscope,the expression of ERS marker GRP78 was determined by immunohistochemistry,and the expression of ERS-related proteins was detected by Western Blot.The results showed that,compared with the control group,in the cadmium group:(1)The cadmium content in testicular tissue was significantly increased(P<0.01)(2)The testicular weight and visceral coefficient had no significant changes,and the epididymis weight and visceral coefficient were both significantly decreased(P<0.05).(3)Sperm motility rate and viability decreased significantly(P<0.01),sperm motility parameters decreased to varying degrees(P<0.05 or P<0.01),and sperm deformity rate increased.(4)The number of sperm cells decreased,the arrangement of spermatogenic cells was disturbed.The wall of the epididymal tubule became thinner.The value of Johnsen score decreased significantly(P<0.01).(5)The secretion level of testosterone decreased significantly(P<0.01).(6)The expression of testosterone synthesis related proteins decreased in different degrees(P<0.05 or P<0.01).(7)The structure of ER is obviously dilated.(8)The positive area of GRP78 increased;the expression of ERS related proteins in testicular tissue is consistent with the increasing trend of ERS.The above results showed that cadmium exposure caused testicular and epididymal injury,spermatogenic function damage,testosterone synthesis disturbance,changes in the structure of ER in testicular Leydig cells.Moreover,cadmium exposure could induce the increased ERS in testicular tissue.2.Role of endoplasmic reticulum stress in cadmium-induced dysfunction of testosterone synthesis in TM3 cellsIn order to explore the effect of ERS on the disturbance of testosterone synthesis induced by cadmium exposure,TM3 cells(mouse testicular Leydig cells)were selected as the object of in vitro study to establish a cadmium poisoning model in vitro.TM3 cells were treated with different concentrations of cadmium(0,5,10,15,20,25,30 μmol/L)for 12 h.The concentration of cadmium was screened by CCK-8.The expression of ERS and testosterone synthesis related protein was detected by Western Blot.The expression of intracellular calcium was detected by flow cytometry.The concentration of ERS inhibitor 4-PBA was screened by CCK-8.Cadmium and 4-PBA were treated alone or in combination,and Western Blot was used to detect the expression changes of proteins related to ERS and testosterone synthesis;ELISA was used to detect the secretion level of testosterone in the cell supernatant.The results showed that:(1)When the concentration of cadmium was 15,20,25,30 μmol/L,the cell viability decreased significantly(P<0.05 or P<0.01).Finally,0,5,10 and 20 μmol/L cadmium were used in the follow-up experiment.(2)Compared with the control group,in the 20μmol/L cadmium group:PERK,ATF6,and IRE1α were activated to varying degrees in TM3 cells(P<0.05 or P<0.01);the expression of GRP78 protein was significantly increased(P<0.01);the intracellular Ca2+ concentration increased significantly(P<0.01).(3)When the concentration of 4-PBA was 40,50,60 umol/L,the cell survival rate decreased significantly(P<0.05 or P<0.01).Finally,30 μmol/L 4-PBA was selected for the follow-up experiment.(4)Compared with the cadmium group,the addition of 4-PBA significantly inhibited the increase of ERS-related protein expression induced by cadmium.(5)Compared with the control group,the expression of testosterone synthesis-related proteins decreased in different degrees in cadmium group(P<0.05 or P<0.01).(6)Compared with cadmium group,the addition of 4-PBA significantly inhibited the decrease of testosterone synthesis protein CYP17A1 and StAR protein expression induced by cadmium.The addition of 4-PBA can alleviate the decrease of testosterone secretion caused by cadmium,but there was no significant difference.The above results indicate that cadmium exposure can cause damage to TM3 cells,leading to enhanced ERS and impaired testosterone synthesis in TM3 cells.ERS plays a promoting role in the disturbance of testosterone synthesis in TM3 cells induced by cadmium.3.The role of endoplasmic reticulum autophagy in cadmium induced disturbance of testosterone synthesis in Leydig cellsIn order to explore the role of ER-phagy in cadmium induced testosterone synthesis disorders,this study used Western Blot to detect autophagy and the expression of ER-phagy related proteins in rat testis and TM3 cells based on cadmium poisoning models in vitro and in vivo.The immunofluorescence was used to detect the LC3 aggregation point of TM3 cells.The ultrastructural changes of endoplasmic reticulum in Leydig cells were observed using transmission electron microscopy.After cells were treated with cadmium alone or in combination with ERS inhibitor 4-PBA,the expression level of ER-phagy related genes was detected using qRT-PCR.The immunofluorescence was used to detect the localization of LC3 and CANX.After cells were treated with cadmium alone or in combination with 4-PBA,the expression of ER-phagy related proteins in TM3 cells was detected by Western Blot.After cells were treated with cadmium alone or in combination with an autophagy inhibitor,chloroquine(CQ),the expression of ER-phagy and testosterone synthesis related proteins in TM3 cells was detected by Western Blot.The results showed that in the in vivo experiment,compared with the control group,the cadmium group:(1)The expression of key autophagy protein LC3 was significantly increased(P<0.01).(2)The expression of FAM134B and TEX264 proteins decreased significantly(P<0.05 or P<0.01).(3)The endoplasmic reticulum was significantly expanded,and a bilayer membrane vesicle that only enclosed fragments of the endoplasmic reticulum was visible.In vitro experiments:(1)Compared with the control group,the expression of LC3 protein in the cadmium group significantly increased(P<0.01),the number of LC3 aggregation points detected by immunofluorescence increased.(2)In the cadmium group,the ER was significantly expanded,and there were bilayer membrane vesicles that only wrapped fragments of the ER.(3)In cadmium group,autophagy key protein LC3 and ER marker protein CANX were co-located.(4)Compared with the control group,the expression of ER-phagy related genes in the cadmium group significantly decreased(P<0.01).Compared with cadmium group,the expression of FAM134B and TEX264 genes in ER-phagy related genes increased significantly after adding 4-PBA(P<0.05 or P<0.01).(5)Compared with the control group,the expression of FAM134B and TEX264 proteins in the cadmium group decreased significantly(P<0.05 or P<0.01).(6)Compared with the cadmium group,the expression of LC3 protein decreased significantly after 4-PBA supplementation(P<0.05),while the expression of FAM134B and TEX264 protein increased significantly(P<0.05).(7)Compared with cadmium group,the expressions of LC3,TEX264 and CYP17A1 were significantly increased after chloroquine was added(P<0.05 or P<0.01).The level of testosterone secretion showed an upward trend,but there was no significant difference.The above results indicate that cadmium exposure can enhance ER-phagy by inducing ERS in Leydig cells.ERS-mediated ER-phagy plays a promoting role in cadmiuminduced testosterone synthesis disorder in Leydig cells.In conclusion,cadmium can cause damage to rat Leydig cells,impaired spermatogenic function,impaired testosterone synthesis,and enhanced ERS.Cadmium exposure can enhance ER-phagy by inducing ERS in Leydig cells.ERS mediated ER-phagy plays a promoting role in cadmium-induced testosterone synthesis disorder in Leydig cells.