The Study Of Distribution Of Zinc In Mouse Epididymis And Effects Of Zinc Deficiency On Epididymal Function In Mice

Objective:Zinc is the second largest trace element necessary for human body.Current studies have shown that zinc is closely related to the male reproductive system of mammals.Epididymis plays an important role in sperm maturation,activation and capacitation.Zinc deficiency may lead to serious reproductive problems,but the location and distribution of zinc in the epididymis and the effect of zinc deficiency on epididymal function are not clear.In this study,a model of zinc deficiency was established to explore the localization and distribution of zinc in the epididymis of mice and the effect of zinc deficiency on the function of epididymis.Methods: 1.The animal model was established with different zinc content diet: zinc normal diet group(zinc:30 mg/kg)and zinc deficiency diet group(zinc:0.85 mg/kg).The body weight,epididymal weight and testicular weight of mice in each group were weighed,and the epididymal index and testicular index were calculated.2.The level of serum testosterone in mice was detected by ELISA.3.Localization and distribution of zinc in mouse epididymis detected by zinc selenide autoradiography(AMG).4.The total zinc concentration of zinc in mouse epididymis was detected by atomic absorption spectrometry(AAS).5.The sperm count,sperm motility and sperm DNA integrity of mouse epididymis were detected.6.Detection of glutathione peroxidase(GSH-Px),superoxide dismutase(SOD),and malondialdehyde(MDA)to explore the antioxidant capacity of epididymis in mice.7.Western blot was used to detect the expression of apoptosis-related protein(Bax,Cyto-C,caspase-9,Caspase 3)and secretion-related protein(CRISP-1,CRISP-4)in mouse epididymis.8.Cell models with different zinc contents were established: normal zinc group,zinc addition group,zinc reduction group,and cell viability was detected by MTT.9.Zinc distribution in MPC-1 cells was detected by zinc fluorescence probe(TSQ)and atomic absorption spectrometry(AAS).10.Western blot was used to detect the expression of apoptosis-related protein(Bax,Cyto-C,caspase 9,Caspase 3)and cysteine-rich secretory proteins(CRISP-1,CRISP-4)in MPC-1 cell.Results: 1.Zinc deficiency diet significantly reduced epididymal index,testicular index and serum testosterone expression level in mice.2.In the normal zinc diet mouse model,zinc-AMG positive particles were mainly concentrated in the epididymal epithelium of the epididymis caput,while almost no zinc-AMG positive particles were detected in the epididymal corpora or the epididymis cauda.Compared with the zinc normal diet group,the zinc-AMG positive particles in the epididymal caput epithelium of the zinc deficiency diet group were significantly decreased,and almost no zinc-AMG positive particles were detected in the epididymal corpora or the epididymis cauda.3.In the epididymis of mice with normal zinc diet,the concentration of total zinc in the epididymis caput was significantly higher than that in the epididymal corpora or the epididymis cauda.4.Zinc deficiency diet significantly reduced epididymal sperm count and sperm motility,increased the sperm fragmentation index(DFI)and the positive rate of spermatozoa A3 in the epididymis of mice.5.Zinc deficiency diet significantly reduced the antioxidant capacity of epididymis in mice.Compared with the mice in the normal zinc diet group,the levels of glutathione peroxidase(GSH-Px)and superoxide dismutase(SOD)in the epididymis of the zinc deficiency group decreased,while the level of malondialdehyde(MDA)increased.6.Zinc deficiency diet significantly increased the expression of pro-apoptotic protein Bax,Cyto-C,Cleaved-caspase-9 and Cleaved-caspase-3,decreased the expression of CRISP-1and CRISP-4 in epididymis.7.TPEN significantly reduced the expression of CRISP-1 and CRISP-4,increased the expression of Bax,Cyto-C,Cleaved-caspase 9,Cleaved-caspase-3 in MPC-1 cell.Conclusion: 1.There was obvious accumulation of zinc in the epididymal epithelium of epididymal caput of mice,and zinc deficiency reduced the accumulation of zinc in the epididymal caput of mice.2.Zinc deficiency decreased the antioxidant capacity of epididymis,decreased the quality of epididymal sperm and caused sperm DNA damage in mice.3.Zinc deficiency increased apoptosis of epididymal epithelium in mice and decreased the expression of cysteine-rich secretory proteins in epididymal epithelium of mice.