Autophagic deficiency is related to steroidogenic decline in aged rat Leydig cells

Abstract

Late-onset hypogonadism (LOH) is closely related to secondary androgen deficiency in aged males, but the mechanism remains unclear. In this study, we found that reduced testosterone production in aged rat Leydig cells is associated with decreased autophagic activity. Primary rat Leydig cells and the TM3 mouse Leydig cell line were used to study the effect of autophagic deficiency on Leydig cell testosterone production. In Leydig cells from young and aged rats, treatment with wortmannin, an autophagy inhibitor, inhibited luteinising hormone (LH)-stimulated steroidogenic acute regulatory (StAR) protein expression and decreased testosterone production. In contrast, treatment with rapamycin, an autophagy activator, enhanced LH-stimulated steroidogenesis in Leydig cells from aged, but not young, rats. Intracellular reactive oxygen species (ROS) levels were increased in both young and aged Leydig cells treated with wortmannin but decreased only in aged Leydig cells treated with rapamycin. Furthermore, an increased level of ROS, induced by H(2)O(2), resulted in LH-stimulated steroidogenic inhibition. Finally, knockdown of Beclin 1 decreased LH-stimulated StAR expression and testosterone production in TM3 mouse Leydig cells, which were associated with increased intracellular ROS level. These results suggested that autophagic deficiency is related to steroidogenic decline in aged rat Leydig cells, which might be influenced by intracellular ROS levels.

Figure 1

Autophagic deficiency occurs in aged rat testes and is associated with the inhibition of StAR expression and decline of serum testosterone. (a) Expression of StAR protein in testes was detected by Western blot analysis. The expression of StAR protein decreased in aged rat testes. (b) Total serum testosterone was lower in aged rats than in young rats. (c) The expression of LC3-II decreased in aged rat testes. (d) Fewer autophagic compartments (including phagophore, autophagosome and autolysosome) were observed in aged rat Leydig cells by EM. The data of the area ratio were not distributed normally. Data are presented as the mean of the area fraction in 50 cells randomly selected from each group. *P<0.05, **P<0.01 (n=10, compared with young rats). Arrow (→), Leydig cells; asterisk (★), swollen mitochondria. Abbreviations: A, autophagosome; AL, autolysosome; EM, electron microscope; IOD, integrated optical density; LC3, microtubule-associated protein light chain 3; M, mitochondria; N, nucleus; StAR, steroidogenic acute regulatory protein.

Figure 2

StAR expression and testosterone production decrease in aged primary rat Leydig cells is associated with autophagic deficiency. (a) The expression of StAR protein was increased by LH stimulation both in young and aged Leydig cells (P<0.01). StAR protein expression in aged Leydig cells was lower than that in young cells both in the presence (P<0.01) and absence (P<0.05) of LH. (b) The supernatant testosterone level was promoted by LH stimulation. The supernatant testosterone level of aged Leydig cells was lower than that of young cells either in the presence or absence of LH (P<0.05). (c) The expression of LC3-II in aged Leydig cells was lower than that in young cells either in the presence (P<0.01) and absence (P<0.05) of LH. (d) The area fraction of punctate GFP-LC3 in young Leydig cells was higher than that in aged ones. Data are presented as the mean of the area fraction in 50 cells randomly selected from each group. *P<0.05, **P<0.01 (n=10). Abbreviations: GFP, green fluorescent protein; IOD, integrated optical density; LC3, microtubule-associated protein light chain 3; LH, luteinizing hormone; StAR, steroidogenic acute regulatory protein.

Figure 3

Alterations of autophagic activity affect LH-stimulated StAR protein expression and testosterone production in primary rat Leydig cells. (a) Effect of autophagic inhibition on LH-stimulated StAR protein expression. For young or aged rat primary Leydig cells, the LH-stimulated StAR protein expression was inhibited by wortmannin, which could be attenuated by NAC. (b) When treated with rapamycin or NAC, the LH-stimulated StAR protein expression increased in aged, but not young, Leydig cells. (c) The effects of autophagic alteration on LH-stimulated testosterone production paralleled that of StAR protein expression. When treated with wortmannin, LH-stimulated testosterone production decreased in both young and aged rat Leydig cells, and this decrease could be attenuated by NAC. (d) Treatment with rapamycin or NAC increased LH-stimulated testosterone production in aged Leydig cells, whereas there was no significant effect on young cells. *P<0.05, **P<0.01 (n=4). Abbreviations: IOD, integrated optical density; LH, luteinizing hormone; NAC, N-acetyl-ℓ-cysteine; Rapa, rapamycin; StAR, steroidogenic acute regulatory protein; WM, wortmannin.

Figure 4

ROS are involved in the changes of LH-stimulated steroidogenesis resulting from autophagic alteration in primary rat Leydig cells. (a) The positive control was treated with 1 mmol l⁻¹ H₂O₂, and the ROS level was strongly increased. Inhibition of autophagy with wortmannin led to increased ROS levels in both young and aged rat Leydig cells, which could be attenuated by NAC. Treatment with NAC or enhancement of autophagic activity with rapamycin led to a decreased ROS level in aged rat Leydig cells but not in young cells. *P<0.05, **P<0.01 (n=4, compared with LH group). (b) Treatment with H₂O₂ led to inhibition of LH-stimulated StAR protein expression and testosterone production in rat primary Leydig cells. *P<0.05, **P<0.01 (n=4, compared with LH group). Abbreviations: IOD, integrated optical density; LH, luteinizing hormone; NAC, N-acetyl-L-cysteine; Rapa, rapamycin; ROS, reactive oxygen species; StAR, steroidogenic acute regulatory protein; WM, wortmannin.

Figure 5

Knockdown of Beclin 1 leads to autophagic deficiency. (a) Knockdown of Beclin 1 led to decreased protein expression of LC3-II. (b) Knockdown of Beclin 1 led to a decreased area fraction of autophagic compartments. (c) Knockdown of Beclin 1 led to a decreased area fraction of GFP-LC3 puncta. **P<0.01 (n=4, compared with NC group). Abbreviations: GFP, green fluorescent protein; IOD, integrated optical density; LC3, microtubule-associated protein light chain 3; LH, luteinizing hormone; NC, negative control; siRNA, small interfering RNA.

Figure 6

Knockdown of Beclin 1 leads to increased cellular ROS levels and down-regulation of LH-stimulated StAR protein expression and testosterone production. (a) LH-stimulated StAR protein expression decreased in TM3 Leydig cells after knockdown of Beclin 1. (b) The LH-stimulated testosterone level decreased in TM3 Leydig cells after knockdown of Beclin 1. (c) The cellular ROS level increased in TM3 Leydig cells after knockdown of Beclin 1. **P<0.01 (n=4, compared with NC group). Abbreviations: LH, luteinizing hormone; NC, negative control; ROS, reactive oxygen species; StAR, steroidogenic acute regulatory protein.