Effects Of High-fat Diet On Testicular Sertoli Cell Junctional Function In Mice And Protective Effects Of Total Saponins From Panax Japonicus

Background Long-term high-fat diet can induce male reproductive dysfunction.Studies have shown that testicular Sertoli cell dysfunction plays an important role in obesity-related male infertility,however its specific molecular mechanism remains to be investigated.Total saponins from Panax Japonicus（TSPJ）are the main active components of Panax japonicus.Our previous studies found that TSPJ can improve reproductive dysfunction of mice induced by high-fat diet,but whether its mechanism is related to the alleviation of Sertoli cell function damage is not clear.Objective The research aims to explore the effect of high-fat diet on testicular Sertoli cell junctional function in mice,as well as the protective effect of total saponins from Panax Japonicus,and explore its possible mechanism.Methods 1.Twelve 6-week-old C57BL/6J male mice were randomly divided into normal diet（ND）group and high-fat diet（HFD）group.The mice in ND group were fed a normal maintenance diet,while the mice in HFD group were fed a high-fat diet.After continuous feeding for 5 months,the following indexes were detected:（1）Using hemocytometer and eosin Y staining,the sperm concentration and viability were assessed.（2）The morphological alterations of testes in mice were observed by hematoxylin-eosin staining,and the seminiferous epithelium height and the seminiferous tubule diameter were measured and counted.（3）The expression levels of apoptosis-related proteins Bax,Bcl2 and cleavage-caspase12 and ER stress-related proteins GRP78,p-PERK,p-e IF2α,p-IRE1 and ATF6αin testes were detected by western blot.The localization and expression of GRP78 in testicular tissue was detected by single-label immunofluorescence.（4）Testicular Sertoli cell nucleus were labeled with SOX9 by single-label immunofluorescence and the number of SOX9 positive cells was counted.（5）The expression levels of tight junction related proteins（ZO-1,Occludin and Claudin11）,ectoplasmic specialization related proteins（N-Cadherin,E-Cadherin andβ-Catenin）and gap junction protein Cx43 in testicular Sertoli cells were detected by western blot.The localization and expression of ZO-1,β-Catenin and Cx43proteins in testicular tissues were detected by single-label immunofluorescence.2.Forty6-week-old C57BL/6J male mice were randomly divided into ND group,HFD group,TSPJ low doses group(TSPJ 25 mg·kg^-1)and high doses group(TSPJ 75 mg·kg^-1),with 10 mice in each group.The mice in ND group were fed a normal maintenance diet,while the mice in the other groups were fed a high-fat diet.The TSPJ-treated groups were respectively given TSPJ for consecutive 5 months by oral gavage at 25 mg·kg^-1and 75 mg·kg^-1,and the other groups were given 5 g·L^-1sodium carboxymethyl cellulose accordingly.After treatment,the mice were weighed,anesthetized and sacrificed.The testes,epididymides,and seminal vesicle tissues were rapidly removed to detect the following parameters:（1）The weights of testes,epididymides and seminal vesicle tissues were measured and their indexes were calculated.（2）The sperm concentration and viability were assessed by hemocytometer and eosin Y staining.（3）The morphological alterations of testes in mice were observed by hematoxylin-eosin staining.The seminiferous epithelium height and the seminiferous tubule diameter were measured and counted.（4）Apoptosis of testicular germ cells of mice was detected by TUNEL staining.（5）The localization and expression of GRP78 in testicular tissue of mice was detected by single-label immunofluorescence.（6）The expression levels of ZO-1,Occludin,N-Cadherin,E-Cadherin andβ-Catenin in testicular tissue were detected by western blot.The localization and expression of ZO-1 andβ-Catenin proteins in testicular tissues were detected by single-label immunofluorescence.（7）The localization and expression of autophagy-related protein LC3B and Akt/m TOR signaling pathway related proteins（p-Akt and p-m TOR）in testicular Sertoli cells of mice were detected by double-label immunofluorescence.Results 1.（1）High-fat diet significantly decreased sperm concentration and sperm viability.（2）High-fat diet induced the exfoliation of spermatogenic cells in testis,loose structure of seminiferous epithelium and significantly decreased the thickness of seminiferous epithelium,but had no significant effect on the diameter of seminiferous tubules.（3）High-fat diet significantly increased the ratio of apoptosis-related protein Bax/Bcl2 expression levels and Cleavage-caspase12 expression levels in testicular tissue.（4）High-fat diet up-regulated the expression of ER stress-related proteins GRP78,ATF6α,p-IRE1,while had no significant effect on the expression of p-PERK and p-e IF2α.（5）High fat diet had no significant effect on the number of testicular Sertoli cells in mice.（6）High fat diet significantly decreased the expression of ZO-1,Occludin,N-Cadherin,E-Cadherin andβ-Catenin proteins,but had no significant effect on the expression level of Claudin11 protein.At the same time,immunofluorescence results showed that high fat diet could significantly decrease the expression of ZO-1 andβ-Catenin proteins in testicular tissue and decrease the continuity,but there was no significant change in expression location.（7）High-fat diet had no significant effect on the expression of Cx43 in testicular Sertoli cells of mice,but immunofluorescence results showed that the expression of Cx43 increased from the base of seminiferous epithelium to the lumen.2.（1）TSPJ could significantly reduce the body weight and increase the index of testis and epididymis in HFD mice,but had no significant effect on the weight of testis,epididymis and seminal vesicle and seminal vesicle index.（2）TSPJ remarkably elevated sperm concentration and viability.（3）TSPJ ameliorated morphological abnormalities of testes and increased seminiferous epithelium height of high-fat diet induced mice,and has no significant effect on the seminiferous tubule diameter.（4）TSPJ could remarkably reduce germ cell apoptosis of HFD mice.（5）TSPJ obviously decreased the expression of GRP78 in testicular tissue of HFD mice.（6）TSPJ effectively up-regulated the expression levels of ZO-1,Occludin,N-Cadherin,E-Cadherin andβ-catenin proteins of HFD mice,and restored the continuity of ZO-1 andβ-Catenin protein expression in testicular tissue.（7）TSPJ down-regulated the expression of LC3B,and up-regulated the expression of p-Akt and p-m TOR proteins in testicular Sertoli cells of HFD mice.Conclusion High-fat diet can induce testicular Sertoli cell junctional function damage in mice,while TSPJ can ameliorate the injury of testicular Sertoli cells junctional function induced by high-fat diet.The mechanism may be related to the inhibition of excessive autophagy in testicular Sertoli cells by activating the Akt/m TOR signaling pathway.