Mild exercise increases dihydrotestosterone in hippocampus providing evidence for androgenic mediation of neurogenesis

Abstract

Mild exercise activates hippocampal neurons through the glutamatergic pathway and also promotes adult hippocampal neurogenesis (AHN). We hypothesized that such exercise could enhance local androgen synthesis and cause AHN because hippocampal steroid synthesis is facilitated by activated neurons via N-methyl-D-aspartate receptors. Here we addressed this question using a mild-intense treadmill running model that has been shown to be a potent AHN stimulator. A mass-spectrometric analysis demonstrated that hippocampal dihydrotestosterone increased significantly, whereas testosterone levels did not increase significantly after 2 wk of treadmill running in both orchidectomized (ORX) and sham castrated (Sham) male rats. Furthermore, analysis of mRNA expression for the two isoforms of 5α-reductases (srd5a1, srd5a2) and for androgen receptor (AR) revealed that both increased in the hippocampus after exercise, even in ORX rats. All rats were injected twice with 5'-bromo-2'deoxyuridine (50 mg/kg body weight, i.p.) on the day before training. Mild exercise significantly increased AHN in both ORX and Sham rats. Moreover, the increase of doublecortin or 5'-bromo-2'deoxyuridine/NeuN-positive cells in ORX rats was blocked by s.c. flutamide, an AR antagonist. It was also found that application of an estrogen receptor antagonist, tamoxifen, did not suppress exercise-induced AHN. These results support the hypothesis that, in male animals, mild exercise enhances hippocampal synthesis of dihydrotestosterone and increases AHN via androgenenic mediation.

Fig. 1.

Experimental design. To exclude acute effects of treadmill running, sample collection was performed 2 d after the last training session. (A and C) ORX or Sham was performed 1 wk before exercise training, at which time the rats were 12 wk old. (B and C) On the day before the 2-wk training began, all of the animals were injected twice i.p. (at 8:00 AM and 8:00 PM) with BrdU (50 mg/kg, B.W.) as a short cell-survival marker in this study. Also, rats were administered the flutamide (30 mg/kg, B.W, s.c.) or the estrogen receptor antagonist tamoxifen (1 mg/kg, B.W., s.c.) suspended in sesame oil 2 h before every exercise session. Rats in the control group were injected with sesame oil only. (D) The enzyme 5α-reductases (Srd5a1,2) convert T into DHT. The effects of T and DHT caused by binding to the AR. T is also converted to estradiol (E2) by the enzyme aromatase (P450arom) and the effect of E2 caused by binding to the estrogen receptors (ERα, β). (E) We have divided adult hippocampal neurogenesis into three primary phases: proliferation, differentiation, and survival of cells. Sham: sham castrated; ORX: orchidectomized; flutamide: an androgen receptor antagonist; tamoxifen: an estrogen receptor antagonist.

Fig. 2.

The effect of treadmill running on hippocampal sex steroidogenesis-related enzymes and steroid receptor mRNA. The mRNA expressions were quantified by real-time PCR. (A, B, and D) Analysis data of mRNA expression for the two isoforms of 5α-reductases (srd5a1: P < 0.0001; srd5a2: P < 0.05), which convert T into DHT, and for ARs (P < 0.01) revealed that both increased in Sham and ORX rats. (C) P450arom, which converts T into E2, mRNA expression was increased in Sham rats only. (E and F) Treadmill exercise increased the mRNA expression of ERα (P < 0.05), but not ERβ, in both Sham and ORX groups. Expressions of mRNA were normalized by GAPDH housekeeping gene as an internal standard. Data represent the mean ± SEM (n = 5–6 rats in each group). *P < 0.05 and ***P < 0.0001 in comparison with sedentary rats (two-way ANOVA). Sham: sham castrated, ORX: orchidectomized.

Fig. 3.

Effects of sex steroid receptor antagonists on exercise-induced AHN of male rats. (A) Mild exercise significantly increased the number of Ki67⁺ cells (proliferation marker) in Veh-, Flu-, and Tam-treated rats (P < 0.0001). (B and C) Flu, but not Tam, blocked the increase of the number of DCX⁺ (immature neuron marker, P < 0.001) and BrdU⁺/NeuN⁺ (cell survival and mature neuron marker, P < 0.01) cells with mild exercise. In immunostaining, the Upper and Lower panels show the cells of exercise rats and sedentary rats. (Left) Veh rats. (Center) Tam-treated rats. (Right) Flu-treated rats. Data represent the mean ± SEM (n = 7–8 rats in each group). *P < 0.05, **P < 0.01 ***P < 0.0001 in comparison with sedentary rats (two-way ANOVA and Bonferroni post hoc tests). Veh: vehicle; Tam: tamoxifen; Flu: flutamide.

Fig. 4.

Effects of orchidectomy on exercise-induced AHN of male rats. (A) Mild exercise significantly increased the number of Ki67⁺ cells in Sham, ORX, and ORX+Flu rats (P < 0.0001). (B and C) Mild exercise increased the number of DCX⁺ and BrdU⁺/NeuN⁺ cells in ORX rats depleted of circulating androgens, and the effects were blocked by Flu. Data represent the mean ± SEM (n = 6–8 rats in each group). *P < 0.05, **P < 0.01, and ***P < 0.0001 in comparison with sedentary rats (two-way ANOVA and Bonferroni post hoc tests). Sham: sham castrated; ORX: orchidectomized; ORX+Flu: orchidectomized+flutamide.