Mechanisms Underlying Inhibitive Effect Of Advanced Glycation End Products On The Testosterone Secretion In Rat Leydig Cells

Recent studies revealed that AGEs(Advanced glycation end products)can be accumulated in the male reproductive tract due to the relatively higher glucose content of the diabete mellitus victims,and combined with receptor of advanced glycation end products(RAGE)expressed in spermatogenic epithelium,epididymis and sperm,and lead to male reproductive dysfunction.The influence of AGEs on testosterone secretion of Leydig cells remains unclear.The present work is aimed to investigate the effects and mechanisms of AGEs on the testosterone secretion in rat leydig cell.The results were reported as follows.(1)The primary leydig cells were isolated and purified from Sprague-Dawley adult male rats.RAGE expresses in rat Leydig cells were analysized by RT-PCR and immunofluorescence.The Leydig cells were treated with human chorionic gonadotropin(h CG)with or without different concentration of AGEs(25,50,100,200 ?g/m L),the medium was collected and testosterone content was assayed by enzyme linked immunosorbent assays(ELISA).The results revealed that RAGE could be expressed in rat Leydig cells,testosterone synthsis could be significantly elevated with h CG addition in the cell medium,and AGEs could remarkably suppress the testosterone production induced by h CG in a dose-dependent manner in rat leydig cells compared with the control group(P< 0.01),and the testosterone production respectively decreased by 26.2%?41.3% and 51.4%.(2)The Leydig cells were exposed to different concentration of AGEs(50,100,200 ?g/m L)and the expression of steroidogenic acute regulatory protein(St AR),3?-hydroxysteroid dehydrogenase(3?-HSD)and cholesterol side-chain cleavage enzyme(P450scc),which involved in testosterone biosynthesis were measured by fluorescence quantitative PCR and Western Blot respectively.The results indicated that the levels of m RNA transcription of the three genes present a downward trend by 12 hours after AGEs treatment.Compared with the control group,the expression of the three genes(St AR,P450 scc and 3?-HSD)of the group exposed to 100 ?g/m L AGEs were significantly decreased by 16.4%,10.4% and 11.6% respectively(P<0.05).After treated with 200 ?g/m L AGEs,the expression of St AR,P450 scc and 3?-HSD decressed significantly(P<0.01).Western-Blot detection results showed that the protein levels of 3?-HSD,P450 scc and St AR of the cells treated by AGEs for 48 h were reduced in a dose-dependent manner compared with the control BSA treatment group in rat leydig cells.Under the treatment with 200 ?g/m L AGEs,St AR,3?-HSD and P450 scc significantly lowered by 42.2%,35.9% and 49.2% respectively complared with control(P<0.01).(3)In order to confirm the inhibitory effects of oxidative stress(OS)and endoplasmic reticulum stress(ERS)caused by AGEs on testosterone secretion of leydig cells,primary leydig cells was pretreated with antioxidant NAC and the ROS content was detected by dichlorofluorescin diacetate(DCFH-DA)probe.The results indicated that NAC could significantly reversed the inhibition of AGEs on the synthesis of testosterone(P<0.01),and AGEs significantly increased the generation of ROS in rat leydig cells in a concentration-dependent manner.Treatment with 200 ?g/m L AGEs significantly enhanced the ROS level complared with control group(P<0.01).These results above declared that AGEs inhibit the generation of testosterone via OS pathway partially.Meanwhile,the Leydig cells were pretreatment with TUDCA,an ERS inhibitor,and the levels of testosterone were measured by ELISA.The result showed that TUDCA could significantly inhibit AGEs-induced decreasing of the testosterone secretion(P<0.01).Western Blot results showed that the expression of ERS related protein CHOP and GRP78 were remarkablely upregulated in a dose-dependent manner.Especially,after treatment with 200 ?g/m L AGEs,the expression quantity of CHOP and GRP78 were respectively 2.40 times and 2.51 times higher than that of control group(P<0.01).It can by inferred that AGEs inhibit the generation of testosterone through ERS partially.(4)To further understanding of the signal pathway involved in the effects of AGEs on testosterone secretion of the leydig cells,ERK1/2 phosphorylation was observed.The result indicated that ERK1/2 phosphorylation of the leydig cells treated by h CG,was significantly increased 3.29 folds than that of the control group.After treated with AGEs,the phosphorylation levels of p-ERK1/2 decreased.Compared with h CG group,the phosphorylation level of p-ERK1/2 of the AGEs groups exposed to 50,100 and 200 ?g/m L decreased by 6.32%,28.8% and 61.6% respectively compared with h CG group(P<0.01).The results above demonstated that ERK1/2 signal transduction pathway invovled in inhibition of testosterone secretion induced by AGEs.In summary,the present study demonstrates that RAGE could be expressed in rat leydig cells and AGEs can inhibit testosterone secretion in rat leydig cells.AGEs-induced the decrease of testosterone production is probably carried out by oxidative stress,endoplasmic reticulum stress and ERK1/2 signal transduction,lowering expression of St AR,3?-HSD,P450 scc in Leydig cells.This study reveals the mechanisms underlying inhibitive effect of AGEs on the testosterone secretion in rat Leydig cells and provides a theoretical basis for male reproductive disorders caused by diabetes.