| ObjectiveTo study the reproductive toxicity of DEHP and the role and mechanism of zinc/JAZF1 in regulating TR4 in DEHP induced spermatogenesis.In this study,Di-2-ethylhexyl phthalate(DEHP)was used as the test substance and pubertal male rats.Methods1、Thirty-two male SD rats were randomly divided into control group and three experimental groups,eight in each,and treated with corn oil for control and DEHP for experimental groups at the doses of 250,500,1 000 mg/kg body weight through gavage,the treatments were conducted once a day for four consecutive weeks.2、After decapitation,the samples were collected,and the changes of Sertoli cells and spermatogenic cells and interstitial cells were examined by flow cytometry method.The apoptotic index of testis cells was detected by TUNEL staining.The sperm motility related indexes were analyzed by computer-aided sperm analysis(CASA).The contents of SOD,MDA and GSH-Px in testis homogenate were measured by ultraviolet spectrophotometry,and the homogenate zinc contents was detected by flame atomic absorption spectrophotometry.The total RNA was extracted from testis tissue and the mRNA expression of related genes such as JAZF1 and TR4 was detected by Real-Time PCR.Results1、Changes in body weight and organ coefficientThe weight of rats in 1 000 mg/kg body weight group decreased significantly as the exposure time prolonged(P<0.05).The testicular weight and Testicular weight and organ coefficient decreased significantly(testicular atrophy),increased liver coefficient(hepatomegaly)and decreased renal coefficient(renal atrophy).2、Sperm motilityCompared with the control group,the sperm density and the linear movement of sperm density in 1 000 mg/kg body weight group decreased significantly(P<0.05),the velocity of straight line,the percentage of A grade sperm,the average swing amplitude,the average beat frequency,the linearity,and the forward movement of sperm in 250 mg/kg body weight group decreased significantly(P<0.05),the linear movement of sperm number decreased(P<0.05)in 250 mg/kg body weight and 1 000 mg/kg body weight groups,the active sperm density in 250 mg/kg body weight and 500 mg/kg body weight groups increased significantly(P<0.05),no significant difference was observed in the percentage of B grade sperm,the percentage of C grade sperm,the percentage of D grade sperm,the velocity of average path,the movement of the swing,the average movement angle and the sperm motility in straight line in each DEHP exposure group3、Testicular histopathologyObservation of HE staining under light microscope can be seen DEHP exposure group can cause varying degrees of testicular seminiferous tubules,spermatogenic cells,Sertoli cells changes damaged.4、The apoptotic index of testis tissueThe apoptotic index of testis tissue in each exposure groups were increased significantly as the dose increases.The abnormal apoptotic cells observed by TUNEL staining were mainly spermatogonia and spermatocytes,and accompanied by a small amount of Sertoli cells.5、Testicular tissue oxidative stressCompared with the control group,the testicular homogenate SOD activity was decreased significantly in each exposure groups(P<0.05);the testicular homogenate MDA activity was increased significantly in each exposure groups(P<0.05);the testicular homogenate GSH-Px in 250mg/kg body weight group decreased significantly(P<0.05).6、Testicular zinc contents and the expression level of JAZF1 and TR4 mRNA in each exposure groups.(1)Compared with the control group,the testicular zinc content in 1000mg/kg body weight group was decreased significantly(P<0.05).(2)Compared with the control group,the expression levels of JAZF1 mRNA in 1000mg/kg body weight group was increased significantly(P<0.05);the expression levels of TR4 mRNA decreased in 1000mg/kg body weight group(P<0.05).Conclusion1、DEHP can lead to testicular atrophy,morphological damage and induced oxidative stress in pubertal rats.2、DEHP can induce apoptosis of testicular cells in pubertal rats.3、The levels of zinc in the testes and the transcriptional levels of JAZF1,TR4 may be associated with DEHP-induced disorders of spermatogenesis.JAZF1 regulates TR4 may be involved in DEHP-induced spermatogenesis. |
