MR 11. Autophagic deficiency may lead to steroidogenic decline in aged Leydig cells

Objective: To explore the role of autophagy in steroidogenic decline of aged Leydig cells.

Methods: Ten young and ten aged male subjects were enrolled in this study. Aging Male's Symptoms (AMS) scale was used for symptom evaluation. The histological changes in testis and ultrastructure of Leydig cells were observed by H.E. staining and electron microscopy (EM) respectively. Steroidogenesis was assessed by of steroidogenic acute regulatory (StAR), protein expression by Western blotting, and serum testosterone detection with an ELISA kit. Autophagic changes were studied via autophagosome analysis in EM and LC3 protein expression analysis by western blot. Beclin 1 was knocked down with siRNA in TM3 mouse Leydig cells followed by StAR and LC3 protein detection and supernatant testosterone measurement. CM-H2DCF-DA, an accepted cell-permeant indicator of ROS, was used to assess the ROS level in Leydig cells.

Results: The scores of AMS in the aged group were s igni f i c ant ly higher than those in the young group (61.25±7.08 vs. 20.75±3.73, P<0.001) with decreased serum testosterone levels [(3.12±0.58) μg/L vs. (6.29±1.17) μg/ L, P<0.05]. HE staining showed degenerative changes in aged human testes. Many swollen mitochondria with mitochondrial cristae disappeared were found in aged human Leydig cells in EM. The expression of StAR protein and serum total testosterone level in aged group were significantly lower than those of young groups. The ratio of autophagic area in EM and LC3-II protein expression were decreased in aged human testes. Knockdown of Beclin 1 decreased LHstimulated StAR expression and testosterone production in TM3 mouse Leydig cells, which were associated with increased intracellular ROS level.

Conclusions: Ageing; autophagy; late onset hypogonadism; leydig cell; reactive oxygen species; testosterone