Functional And Mechanistical Investigation Of The Whitening Adipose Tissues-Induced By High-Fat Diet On The Spermatogenic Environment Of Mice

OBJECTIVE:To study the effects of obesity induced by high-fat diet on the spermatogenic environment of mice and interrogate the mechanism accounting for the crosstalk between adipose tissues and the diverse cell types involved in spermatogenesis.METHODS:A high-fat diet-induced obese mouse model was established.H&E staining was used to detect the effect of obesity on mouse testis morphology.ELISA was used to detect testosterone and secretion of AMH,GDNF and INH in obese mice.mRNA differential sequencing was used to find differential genes in adipose tissue between obese mice and control mice.Differentially expressed genes were determined by methods such as qPCR.ELISA and IHC.At the cellular level,the function of differentially expressed genes was verified by qPCR,WB,ELISA methods.The interaction receptors of differentially expressed genes were determined by immunoprecipitation and immunofluorescence,and verified by si-RNA and small molecule inhibitors.At the in vivo level,the differential gene protein factor was injected into the mouse testis by microinjection of the testicular seminiferous tubules to examine its effect on mouse testicular function.RESULTS:Upon 12 weeks of high-fat diet induction,we found that the obese mice gained weight,and the serum levels of insulin,blood sugar,blood lipids and other biochemical indicators were increased.The serum levels of several inflammatory factors were also augmented.Furthermore,reduced testicular volume and sperm counts were observed.The diameter of the seminiferous tubules in the testis of obese mice was reduced as shown by H&E staining,and the thickness of the seminiferous epithelium was thinned.Moreover,the levels of serum GnRH,LH,testosterone,and AMH,GDNF,and INH were decreased in obese mice,and estradiol levels were elevated as indicated by the results of ELISA.Quantitative PCR and WB results revealed that the expression levels of key genes and transcription factors correlated with testosterone biogenesis were decreased,for instance,P450scc,P450c17,StAR and Nur77.We also found that the expression levels of Amh,Gdnf and Inha genes were decreased in obese mice.mRNA sequencing of adipose tissues showed a significant decrease in the expression levels of clusterin and resistin in eWAT and iWAT adipose tissue of obese mice compared with that in control mice.By The clusterin and resistin were validated as adipocytokines secreted by adipose tissue as shown by qPCR,ELISA analyses Next,TM3 and TM4 cells were treated with the two factors as mentioned above,respectively.Quantitative PCR and WB results showed that both the two factors inhibited the expression of testosterone genes P450scc,P450c17,StAR and Nur77 and the secretion of testosterone in TM3 cells.Meanwhile,these two factors also inhibited the expression of Amh,Gdnf,Inha genes and protein secretion in TM4 cells.The results of immunoprecipitation and immunofluorescence showed that VLDLR and TLR4 are receptors for clusterin and resistin in TM3 and TM4 cells,respectively.The microinjection of clusterin and resistin testicular seminiferous tubules showed that under in vivo conditions,the injection of clusterin and resistin resulted in a decrease in testicular volume in mice,and the H&E results showed that the spermatogenic epithelial cells of the testicular seminiferous tubules were severely detached.At the same time,by quantitative PCR,the results of WB showed that the expression of P450scc,P450c17,StAR,Nur77 and Amh,Gdnf,Inha,and Jam,Zo-1,Cldn11 and Ocln were decreased in the testis.The ELISA results showed the secretion of testosterone and AMH,GDNF and INH cut down.CONCLUSION:Our findings indicate that the testes of obese mice induced by high-fat diet decreased and the number of sperm decreased.High-fat-induced white adipose tissue inhibits the secretion of testosterone in mouse Leydig cells and the secretion of AMH,GDNF,and INH in Sertoli cell by secreting clusterin and resistin,thereby destroying the spermatogenic environment of mouse testis,resulting in the number of testes and sperm cut back.