| Objective:In this study,the animal model of obese male rats was established and treated with zinc sulfate.By detecting the expression and localization of zinc transporters(ZIP1,ZIP2,ZNT1 and ZNT8)and apoptosis-related proteins(BCL-2,BAX,Caspase-9 and Caspase-12)in testicular tissue,the aim of this study was to explore the effect and mechanism of zinc sulfate on reproductive injury in obese rats.Methods:After one week of adaptive feeding,30 SD male rats(5-6weeks old)were randomly divided into 2 groups:control group(n=8,normal diet,ND)and obesity group(n=22,high fat diet,HFD).Weight was measured at the same time every week.After 8 weeks,16 obese rats were successfully established and 6 failed.Sixteen successful obese rats were randomly divided into two groups:obesity model group(HFD,n=8)and zinc sulfate group(HFD+Zn SO4,n=8).Zinc sulfate 25mg/kg body weight was given daily to the HFD+Zn SO4 group,and the same amount of normal saline was given to the ND group and the HFD group.After 8 weeks,the bilateral testes and peri-testicular fat of rats were weighed,and the fresh epididymal tail spermatozoa were taken to detect the concentration,motility,DFI and CMA3;determination of serum CHO,TG,LDL,HDL,GLU,T,E2,PRL;determination of testicular and prostate tissue zinc content;the morphological changes of testicular tissue were observed by HE staining;the localization of zinc transport protein and apoptosis-related protein in testicular tissue was observed by immunohistochemical method;the m RNA and protein expressions of zinc transporter and apoptosis-related proteins in testis were detected by quantitative real-time PCR and Western Blot.Results:1. Comparison of general conditions of ratsCompared with the ND group,the body weight,peritesticular fat weight and Lee's index in the HFD group were significantly increased(P<0.05).Compared with the HFD group,the body weight,peritesticular fat weight and Lee's index in the HFD+Zn SO4 group were significantly decreased(P<0.05).There was no significant difference in body length and bilateral testicular weight among groups(P>0.05).2. Comparison of sperm parameters of ratsCompared with the ND group,the sperm concentration and motility in the HFD group were significantly decreased(P<0.05),DFI and CMA3~+was significantly increased(P<0.05).Compared with the HFD group,the sperm concentration and motility in the HFD+Zn SO4 group were significantly increased(P<0.05),DFI and CMA3~+was significantly decreased(P<0.05).3. Comparison of serum indexes of rats3.1 Comparison of serum biochemical indexes of ratsCompared with the ND group,the levels of serum CHO,TG and LDL in the HFD group were significantly increased(P<0.05),HDL was significantly decreased(P<0.05).Compared with the HFD group,the levels of serum CHO,TG and LDL in the HFD+Zn SO4 group were significantly decreased(P<0.05),HDL was significantly increased(P<0.05).There was no significant difference in blood glucose among the three groups(P>0.05).3.2 Comparison of serum sex hormone indexes of ratsCompared with the ND group,the serum T in the HFD group was significantly decreased(P<0.05).Compared with the HFD group,the serum T in the HFD+Zn SO4 group was significantly increased(P<0.05).There was no significant difference in E2 and PRL among the groups(P>0.05).4. Comparison of zinc content in testis and prostate tissue of ratsCompared with the ND group,the content of zinc in testis and prostate in HFD group was significantly decreased(P<0.05).Compared with the HFD group,the content of zinc in testis and prostate in HFD+Zn SO4 group was significantly increased(P<0.05).5. Histopathological changes of testisHE staining showed that in the ND group,the testicular seminiferous tubules were neatly arranged,there were a large number of interstitial cells between the tubules,and there were multiple layers of spermatogenic cells and Sertoli cells in the lumen.The cells were arranged tightly,clearly and structurally,and a large number of mature sperm could be seen in the lumen.In the HFD group,the arrangement of seminiferous tubules was loose,the spacing became larger,the interstitial cells decreased,the structure of the basal layer of seminiferous tubules was incomplete,the spermatogenic cells and Sertoli cells at all levels decreased and vacuoles appeared,and the sperm in the lumen decreased greatly.In the HFD+Zn SO4 group,the seminiferous tubules were arranged neatly,tightly and slightly apart,and all levels of spermatogenic cells and Sertoli cells could be seen in the lumen,but the number of spermatogenic cells was less than that of the ND group,the arrangement of cells was more orderly,and the number of spermatozoa in the lumen.6. Localization of apoptosis-related proteins and zinc transporters in the testis of rats in each group6.1 Localization of zinc transporter in the testis of rats in each groupIHC results showed that ZIP1,ZIP2 and ZNT1 were mainly expressed in all levels of spermatogenic cell membrane and cytoplasm,while ZNT8 was mainly expressed in spermatogonia at the base of seminiferous tubules and interstitial Leydig cells.6.2 Localization of apoptosis-related proteins in the testes of rats in each groupIHC results showed that BCL-2,BAX,Caspase-9 and Caspase-12 was mainly expressed in the cytoplasm of seminiferous cells and interstitial cells.7. The m RNA expression of apoptosis-related protein and zinc transporter in testis of rats in different groups7.1 The m RNA expression of zinc transporter in the testis of rats in each group.Compared with the ND group,the m RNA expression of ZIP1(P<0.01)and ZIP2(P<0.05)were decreased,the m RNA expression of ZNTI(P<0.01)and ZNT8(P<0.05)were increased in the HFD group.Compared with the HFD group,the m RNA expression of ZIP1(P<0.01)and ZIP2(P<0.05)were increased,the m RNA expression of ZNT1(P<0.01)and ZNT8(P<0.05)were decreased in the HFD+Zn SO4 group.7.2 The m RNA expression of apoptosis-related proteins in testes of rats in different groups.Compared with the ND group,the m RNA expression of BCL-2(P<0.01)were decreased,the m RNA expression of BAX(P<0.01),Caspase-9(P<0.01)and Caspase-12(P<0.05)were increased in the HFD group.Co-mpared with the HFD group,the m RNA expression of BCL-2(P<0.01)were increased,the m RNA expression of BAX(P<0.01),Caspase-9(P<0.05)and Caspase-12(P<0.01)were decreased in the HFD+Zn SO4 group.8. The protein expression of apoptosis-related protein and zinc transporter in testis of rats in each group.8.1 The protein expression of zinc transporter in testis of rats in each group.Compared with the ND group,the protein expression of ZIP1decreased(P<0.05),the protein expression of ZNT1 and ZNT8increased(P<0.05),but the expression of ZIP2 protein had no significant difference in the HFD group(P>0.05).Compared with the HFD group,the expression of ZIP1 increased(P<0.05),the protein expression of ZNT1 and ZNT8 decreased(P<0.05),and the expression of ZIP2 protein had no significant difference in HFD+Zn SO4 group(P>0.05).8.2 The protein expression of apoptosis-related in testes of rats in each group.Compared with the ND group,the protein expression of BCL-2(P<0.01)were decreased,the protein expression of BAX,Caspase-9 and Caspase-12 were increased in the HFD group(P<0.01).Compared with the HFD group,the protein expression of BCL-2(P<0.01)were increased,the protein expression of BAX,Caspase-9 and Caspase-12 were decreased in the HFD+Zn SO4 group(P<0.01).Conclusions:1.The obese male rats showed the decrease of semen quality,the decrease of zinc content in testis and prostate,the destruction of testicular tissue structure,the disordered expression of zinc transporter,the overexpression of apoptotic protein and so on,suggesting that obesity can damage male reproductive function.2.After zinc sulfate treatment,the obese male rats showed the increase of semen quality,the increase of zinc content in testis and prostate,the testicular tissue structure recovered,the expression of zinc transporter and apoptotic protein basically returned to normal,suggesting that zinc sulfate may improve male reproductive function damage caused by obesity by affecting zinc transport protein and apoptosis-related protein. |
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