| Background Great attention has been paid to the research of aging or aging-related diseases with the increasing aging population.Clinical data indicated aging can lead to a reduced serum testosterone level,including hopogonadism or other disease.Object To investigate the oxidative stress,endoplasmic reticulum stress and possible molecular mechanism in testis and epididymal adipose tissue the role in age-related testosterone levels decresed.Methods C57BL/6J male mice of different age groups were purchased from the animal center at Anhui Medical University,and further divided into three groups:(1)3months(young group,n=8),(2)12 months(middle-aged group,n=8),(3)24 months(aged group,n=8).Mice were sacrificed by cervical dislocation and dissected to weigh the testis and epididymal adipose tissue;testicular tissue was collected for paraffin section,F4/80 marked macrophage infiltration and 3β-HSD expression were evaluated by immunohistochemical staining;Western blotting was applied to detect NF-k B,GRP78 and antioxidant GPx4,Mn SOD in the epididymal adipose tissue,and steroidogenic enzyme in testicular tissue,endoplasmic reticulum stress moleculer GRP78,SIRT1,and the expression levels of antioxidant Mn SOD,GPx4;Real-time RT-PCR was utilized to detect the expressions of steroidogenic m RNA in testicular tissue,as well as antioxidase and endoplasmic reticulum stress markers.Results1.Histological changes Via anti-3 β-HSD monoclonal antibody marking,no significant difference was observed in Leydig cell numbers of three groups,while 3β-HSD expression in aged group was significantly lower than young group by average optical density from image analysis software IPP6.0(p<0.01).Macrophage numbers in testicular tissue of aged group was evidently higher than that of young group according to macrophage specific antibody F4/80 by immunohistochemical staining(p<0.01).2.Reduced steroidogenic machinery during aging In testicular tissues,St AR and P450 scc expression levels with their m RNA expression were significantly lower in middle-aged group than young group(P<0.05),and continuously decreased in aged group.Cyp17 a level was lower in middle-aged group than young group,and significantly reduced in aged group(P<0.05).Hsd17 b level in middle-aged group and aged group were remarkably decreased than young group(P<0.05).3.Age-associated disruption of antioxidant status in testicular In testicular tissues,SIRT1 protein expression was lowered in middle-aged group comparing to young group,and significant decrease was observed in aged group comparing to middle-aged group(P<0.05).By comparison with young group,Sirt1 m RNA levels in aged and middle group were noticeably reduced(P<0.05).Mn SOD and GPx4 were lower in middle-aged group than young group(P<0.05),with continuously decreased level in aged group(P<0.05).Significantly lower levels of Sod2 m RNA and Gpx4 m RNA were found in aged and middle-aged group than young grou(P<0.05).4.ER stress markers increase in the testicular tissues of the aged mice In testicular tissues,GRP78 protein expression was significantly elevated in middle-aged group when compared to young group,with a more elevated level in aged group(P<0.05).The similar tendency was gained as Grp78 and Chop m RNA levels.5.Antioxidant status decrease,ER stress marker and proinflammatory transcription factor increase in the epididymal adipose tissues of the aged mice In mice epididymal adipose tissues,Mn SOD and GPx4 protein decrease in aged group compared with young mice.GRP78 and NF-k B protein expression in aged group was remarkably higher than that of young group NF-k B expression level was significantly higher in aged group than young group(P<0.05).Conclusion The results indicated aging related oxidative stress,endoplasmic reticulum stress and inflammation in testicular and epididymal adipose tissue could cause reduced testosterone synthesis related proteins/enzymes levels and reduced testosterone level. |
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