Testosterone replacement in transgenic sickle cell mice controls priapic activity and upregulates PDE5 expression and eNOS activity in the penis

Abstract

Sickle cell disease (SCD)-associated priapism is characterized by decreased nitric oxide (NO) signaling and downregulated phosphodiesterase (PDE)5 protein expression and activity in the penis. Priapism is also associated with testosterone deficiency, but molecular mechanisms underlying testosterone effects in the penis in SCD are not known. Given the critical role of androgens in erection physiology and NO synthase (NOS)/PDE5 expression, we hypothesized that testosterone replacement to eugonadal testosterone levels reduces priapism by reversing impaired endothelial (e)NOS activity and molecular abnormalities involving PDE5. Adult male transgenic Berkeley sickle cell (Sickle) and wild-type (WT) mice were implanted with testosterone pellets, which release 1.2 μg testosterone/day for 21 days, or vehicle. After 21 days, animals underwent erectile function assessment followed by collection of blood for serum testosterone measurements, penes for molecular analysis, and seminal vesicles as testosterone-responsive tissue. Serum testosterone levels were measured by radioimmunoassay; protein expressions of PDE5, α-smooth muscle actin, eNOS and nNOS, and phosphorylation of PDE5 at Ser-92, eNOS at Ser-1177, neuronal (n) NOS at Ser-1412, and Akt at Ser-473 were measured by Western blot in penile tissue. Testosterone treatment reversed downregulated serum testosterone levels and increased (p < 0.05) the weight of seminal vesicles in Sickle mice to levels comparable to that of WT mice, indicating restored testosterone levels in Sickle mice. Testosterone treatment reduced (p < 0.05) prolonged detumescence in Sickle mice and normalized downregulated P-PDE5 (Ser-92), PDE5, P-eNOS (Ser-1177), and P-Akt (Ser-473) protein expressions in the Sickle mouse penis. Testosterone treatment did not affect P-nNOS (Ser-1412), eNOS, nNOS, or α-smooth muscle actin protein expressions in the Sickle mouse penis. In conclusion, in the mouse model of human SCD, increasing testosterone to eugonadal levels reduced priapic activity and reversed impaired Akt/eNOS activity and PDE5 protein expression in the penis.

Keywords: Akt; eNOS phosphorylation; nNOS phosphorylation.

Figure 1

Testosterone treatment restores serum testosterone levels (A) and seminal vesicles weight (B) in Sickle mice. Mice were implanted with testosterone pellets (0.025 mg/pellet, which release 1.2 μg testosterone/day for 21 days) or vehicle pellets. After 21 days, blood and penis were collected for testosterone measurements and molecular analysis, respectively, and seminal vesicle weight was recorded. Each bar represents the mean ± SEM. *, P < 0.05 vs. WT + vehicle; #, P < 0.05 vs. Sickle + vehicle; +, P < 0.05 vs. WT + testosterone. n = 15/treatment. Veh = vehicle; T = testosterone.

Figure 2

Testosterone treatment reduces prolonged detumescence in Sickle mice. 3 weeks following testosterone or vehicle pellet implantation, electrical stimulation of the cavernous nerve was performed as voltage response (0.3, 1, 2, and 4 volts) for 1 minute to measure erectile function. Erectile response to electrical stimulation of the cavernous nerve is indicated by maximal ICP/mean arterial pressure (MAP, A), total ICP/MAP (B), detumescence time (C), and detumescence area (D). Each bar represents the mean ± SEM. *, P < 0.05 vs. WT + vehicle; ^#, P < 0.05 vs. Sickle + vehicle. n = 5/treatment. AU = arbitrary units. Veh = vehicle; T = testosterone.

Figure 3

Testosterone treatment restores P-PDE5 (Ser-92) (A) and PDE5 (B) protein expressions in the sickle mouse penis. Upper panels are representative Western immunoblots of P-PDE5 (Ser-92), PDE5, and GAPDH in penes of WT-vehicle, WT-testosterone, Sickle-vehicle, and Sickle-testosterone treated mice. Lower panels represent quantitative analyses of P-PDE5/GAPDH and PDE5/GAPDH in penes in the same treatment groups. Each bar represents the mean ± SEM. *, P < 0.05 vs. WT + vehicle; ^#, P < 0.05 vs. Sickle + vehicle. n = 6/treatment. Veh = vehicle; T = testosterone.

Figure 4

Testosterone treatment does not affect α-SMA protein expression in the WT or Sickle mouse penis. Upper panels are representative Western immunoblots of α-SMA and GAPDH in penes of WT-vehicle, WT-testosterone, Sickle-vehicle, and Sickle-testosterone treated mice. Lower panel represents quantitative analysis of α-SMA/GAPDH in penes in the same treatment groups. Each bar represents the mean ± SEM. n = 6/treatment. Veh = vehicle; T = testosterone.

Figure 5

Testosterone treatment restores P-eNOS (Ser-1177) (A) and P-Akt (Ser-473) (B) expressions, and does not affect eNOS protein expression (C), in the Sickle mouse penis. Upper panels are representative Western immunoblots of P-eNOS (Ser-1177), eNOS, P-Akt, Akt, and GAPDH in penes of WT-vehicle, WT-testosterone, Sickle-vehicle, and Sickle-testosterone treated mice. Lower panels represent quantitative analysis of P-eNOS (Ser-1177)/eNOS, P-Akt/Akt, and eNOS/GAPDH in penes in the same treatment groups. Each bar represents the mean ± SEM. *, P < 0.05 vs. WT + vehicle; ^#P < 0.05 vs. Sickle + vehicle. n = 6-7/treatment. Veh = vehicle; T = testosterone.

Figure 6

Testosterone treatment does not affect P-nNOS (Ser-1412) (A) and nNOS (B) protein expressions in the Sickle mouse penis. Upper panels are representative Western immunoblots of P-nNOS (Ser-1412), nNOS, and GAPDH in penes of WT-vehicle, WT-testosterone, Sickle-vehicle, and Sickle-testosterone treated mice. Lower panels represent quantitative analysis of P-nNOS (Ser-1412)/nNOS and nNOS/GAPDH in penes in the same treatment groups. Each bar represents the mean ± SEM. n = 6-7/treatment. Veh = vehicle; T = testosterone.