| Objective:In this study,an animal model of oligoasthenospermia rats was constructed and zinc sulfate intervention was carried out.By analyzing the dynamic changes of zinc homeostasis and detecting the expression and localization of oxidative stress pathway-related proteins(NRF2,KEAP1,HO-1,HDAC2)and testosterone production-related regulatory proteins(LHR,St AR,CYP11A1,HSD3β1)in testicular tissues,aiming to explore the effect and mechanism of zinc supplementation on the reproductive function of oligoasthenospermia rats.Methods:After one week of adaptive feeding,23 male SD rats(SPF grade,6 weeks old)were randomly divided into three groups: 7 in the control group(control,C),9 in the model group(model,M)and 7 in the zinc sulfate group(Zn SO4).Rats in all groups were fed with normal diet,and the body weight was measured every Monday morning.The model group and the Zn SO4 group was given tripterygium glycosides(40 mg/kg/d)by gavage to build the model,the control group was treated with the same amount of normal saline at 8:00-10:00 every morning.At the end of the 4th week,two rats in the M group were randomly dissected to determine whether the oligoasthenospermia model was successful,and 14 rats were finally successful.Starting from the 5th week,after intragastric administration of tripterygium glycosides for 4 hours,the Zn SO4 group was given Zn SO4(25mg/kg/d)gavage for protective treatment for 4 weeks.Meanwhile,the control group and the model group were given the same amount of normal saline for second gastric gavage.Finally,7 rats in the C group,7 rats in the M group,and 7 rats in the Zn SO4 group.After 8 weeks,the rat’s bilateral testes and epididymis were weighed separately,and fresh epididymal tail sperm was taken to detect its concentration and viability;serum Zn,T and tissue Zn were measured;the content of oxidative stress-related proteins and zinc-containing enzymes in testicular tissue was detected by ELISA;the histopathological changes of testis was observed by HE staining;the location of testosterone production-related regulatory proteins and oxidative stress-related proteins in testis tissue was observed by immunohistochemistry;the testosterone production-related regulatory proteins and oxidative stress-related proteins m RNA and protein expression in testicular tissue was detected by q PCR and Western Blotting.Results: 1.Comparison of general conditions of rats There was no statistical difference in body weight,body length,average weight of both testes and epididymis in each group(P>0.05).2.Comparison of sperm parameters of rats Compared with the C group,the sperm concentration and vitality of both M group and Zn SO4 group were significantly reduced(P<0.05);compared with the M group,the sperm concentration and vitality of Zn SO4 group was significantly increased(P<0.05).3.Comparison of the concentrations of serum zinc,testosterone and tissue zinc of rats Compared with the C group,the concentration of serum zinc,testosterone and tissue zinc in the M group was significantly reduced(P<0.05),and the serum testosterone level in Zn SO4 group increased apparently(P<0.05),there was no significant difference in concentration of serum zinc and tissue zinc in Zn SO4 group(P>0.05).Compared with the M group,the concentration of serum zinc,testosterone and tissue zinc in Zn SO4 group increased apparently(P<0.05).4.Comparison of enzyme content in testis tissues of rats ⑴ Comparison of the content of oxidative stress-related enzymes in rat testis Compared with the C group,T-AOC,SOD and GSH-PX of the testis tissue of the rats in the M group were significantly reduced(P<0.05),and the MDA was significantly increased(P<0.05).There was no statistically significant difference among T-AOC,SOD,GSH-PX and MDA in the testis tissue of rats in the Zn SO4 group(P>0.05);compared with the M group,the T-AOC,SOD and GSH-PX of the testis of the rats in the Zn SO4 group was significantly increased(P<0.05),and the MDA decreased(P<0.05).⑵ Comparison of zinc enzyme content in rat testis Compared with the C group,ALP,LDH,ADH,and ATP in the testis of the M group were significantly reduced(P<0.05),and there was no significant difference in ALP,LDH,ADH,and ATP in the testis of the Zn SO4 group(P>0.05);Compared with the M group,the the concentration of ALP,LDH,ADH and ATP in the testis tissue of the Zn SO4 group increased significantly(P<0.05).5.Histopathological changes of rat testis The results of HE staining showed that the seminiferous tubules in the C group were arranged neatly,there were a large number of Leydig cells between the tubes,and there were multiple layers of spermatogenic cells at various developmental stages in the lumen,and Sertoli cells were also seen.The arrangement of various cells was dense,clear and structural,and a large number of mature sperm can be seen in the lumen.while the seminiferous tubules in the M group are arranged disorderly,the distance between the tubes is large,the Leydig cells are reduced,the basal structure of the tubules is incomplete.In addition,the lumen is enlarged,all levels of spermatogenic cells and Sertoli cells were significantly reduced and vacuoles increased,and sperm in the lumen was significantly reduced.In the Zn SO4 group the seminiferous tubules were arranged more neatly and tightly but slightly spaced.Sertoli cells and spermatogenic cells at various developmental stages were seen in the lumen,while the number was less than that in the C group,but increased compared with the M group.the arrangement of spermatogenic cells at all levels was more orderly,and the number of sperm in the lumen increased significantly compared with the M group.6.Localization of oxidative stress-related proteins and testosterone production-related regulatory proteins in the testes of rats in each group ⑴ Localization of oxidative stress-related proteins in the testes of rats in each group IHC results show that NRF2 is mainly expressed in spermatogenic cells at all levels;KEAP1 is strongly expressed in Laydig cells,Sertoli cells and sperm,as well as in spermatogenic cells at all levels;HO-1 is mainly expressed in Sertoli cells;HDAC2 is mainly expressed in Spermatogonia at the base of the seminiferous tubules..⑵ Localization of apoptosis-related proteins in the testes of rats in each group IHC results showed that LHR was expressed intensively in Leydig cells,St AR,CYP11A1 and HSD3β1 were mainly expressed in Leydig cells,while sperm and spermatogonia at the base of seminiferous tubules were also can be seen.7.The m RNA expression of oxidative stress-related proteins and testosterone production-related regulatory proteins in the testes of rats in different groups ⑴ The m RNA expression of oxidative stress-related proteins in the testes of rats in each group Compared with the C group,the m RNA expression of NRF2(P<0.05),KEAP1(P<0.05)and HO-1(P<0.05)in the testis of the M group decreased,and the difference in the m RNA expression of HDAC2 was not statistically significant(P>0.05);There was no significant difference in the expression of NRF2,KEAP1,HO-1 and HDAC2 m RNA in the testis of the Zn SO4 group(P>0.05).Compared with the M group,the m RNA expressions of NRF2(P<0.05),KEAP1(P<0.05)and HO-1(P<0.05)in the testis of the Zn SO4 group increased,but the m RNA expression of HDAC2 was not significantly different(P>0.05).⑵ The m RNA expression of testosterone production-related regulatory proteins in the testes of rats in each group Compared with the C group,the m RNA expression of LHR(P<0.05),StAR(P<0.05),CYP11A1(P<0.05)and HSD3β1(P<0.05)in the testis of the M group was reduced;There was no significant difference in the m RNA expression of LHR,St AR,CYP11A1 and HSD3β1 in the testis of the Zn SO4 group(P>0.05).Compared with the M group,the m RNA expression of LHR(P<0.05),St AR(P<0.05),CYP11A1(P<0.05)and HSD3β1(P<0.05)in the testis of the Zn SO4 group increased.⑶ The m RNA expression of zinc transporters Zn T4,Zn T8,ZIP1 and ZIP6 in the testes of rats in each group Compared with the C group,the m RNA expression of Zn T4(P<0.05),Zn T8(P<0.05)and ZIP6(P<0.05)in the testis of the M group was reduced,and the m RNA expression of ZIP1(P>0.05)had no significant difference;There was no significant difference in the m RNA expression of Zn T4,Zn T8,ZIP1 and ZIP6 in the Zn SO4 group(P>0.05).Compared with the M group,the m RNA expression of Zn T4(P<0.05),Zn T8(P<0.05)and ZIP6(P<0.05)increased in the Zn SO4 group,and the difference in the m RNA expression of ZIP1(P>0.05)was not statistically significant.8.The protein expression of oxidative stress-related proteins and testosterone production-related regulatory proteins in the testes of rats in each group ⑴ The protein expression of oxidative stress-related proteins in testes of rats in each group Compared with the C group,the protein expression of NRF2(P<0.05),HO-1(P<0.05),KEAP1(P<0.05)in the testis of M group decreased,and the protein expression of HDAC2 increased(P<0.05);In the Zn SO4 group,the protein expression of NRF2 and HO-1 in the testes decreased(P<0.05).There was no statistically significant difference in protein expression between KEAP1 and HDAC2(P>0.05).Compared with the M group,the protein expressions of NRF2(P<0.05),HO-1(P<0.05)and KEAP1(P<0.05)in the testis of the Zn SO4 group increased,but the expression of HDAC2 protein had no statistically significant difference in the Zn SO4 group(P>0.05).⑵ The protein expression of apoptosis-related proteins in testes of rats in each group Compared with the C group,the expression of LHR,St AR,CYP11A1 and HSD3β1 protein in the M group decreased significantly(P<0.05);and the expression of LHR,CYP11A1 and HSD3β1 protein in the testis of Zn SO4 group increased(P<0.05),There was no statistically significant difference in St AR protein expression(P>0.05).Compared with the M group,the expressions of LHR,St AR,CYP11A1 and HSD3β1 proteins in the Zn SO4 group were significantly increased(P<0.05).Conclusions: After zinc sulfate treatment,the semen quality of oligoasthenospermia rats increased,the testicular tissue structure recovered,the contents of serum zinc,testicular tissue zinc and zinc containing enzymes increased,the expression of proteins related to oxidative stress pathway increased and the content of antioxidant enzymes in testicular tissue increased,the expression of testosterone production related proteins basically returned to normal.These results suggest that zinc sulfate may improve the male reproductive function of oligoasthenospermia rats by repairing the destroyed zinc homeostasis,activating KEAP1-NRF2/ARE signaling pathway and the synthesis of downstream antioxidants,regulating the function of testosterone production related enzymes. |
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