Reproductive Endocrinology and Signaling Group, Department of Biology and Ecology, Faculty of Sciences, University of Novi Sad, Novi Sad, Serbia.
Biol Reprod. 2010 Sep;83(3):434-42. doi: 10.1095/biolreprod.110.083626. Epub 2010 May 12.
Testosterone, acting as a systemic and local factor, is one of the major regulatory molecules that initiate and maintain testicular function. In the present study, different experimental approaches were used to evaluate the role of testosterone in regulation of the nitric oxide (NO)-cGMP pathway in Leydig cells derived from normal and hypogonadotropic male rats treated with testosterone for 24 h and 2 wk. Real-time quantitative PCR and Western blot analysis revealed increased inducible NO synthase (NOS2) expression followed by increased NO secretion from Leydig cells ex vivo after continuous treatment with testosterone for 2 wk in vivo. The cGMP-specific phosphodiesterases Pde5, Pde6, and Pde9 were up-regulated, whereas PRKG1 protein was decreased after a 2-wk testosterone treatment. Induction of Nos2 and Pde5 in Leydig cells was blocked by androgen receptor antagonist. In experimental hypogonadotropic hypogonadism, expression of NOS2 was significantly reduced, and treatment with testosterone increased NOS2 expression above control levels. PDE5 protein level was unchanged in hypogonadal rats, whereas treatment of hypogonadal rats with testosterone significantly increased it. In contrast, hypogonadism and testosterone replacement reduced PRKG1 protein in Leydig cells. In vitro treatment with testosterone caused gradually increased Nos2 gene expression followed by increased nitrite and cGMP production by purified Leydig cells. In summary, testosterone up-regulated NO signaling via increased NOS2 expression and contributed to down-regulation of cGMP signaling in Leydig cells. Thus, testosterone-induced modulation of NO-cGMP signaling may serve as a potent autocrine regulator of testicular steroidogenesis.
睾酮作为一种全身性和局部性因子，是启动和维持睾丸功能的主要调节分子之一。在本研究中，使用了不同的实验方法来评估睾酮在调节正常和促性腺激素低下雄性大鼠睾丸间质细胞中一氧化氮（NO）-cGMP 通路中的作用，这些大鼠经过 24 小时和 2 周的睾酮处理。实时定量 PCR 和 Western blot 分析显示，体内连续 2 周用睾酮处理后，诱导型一氧化氮合酶（NOS2）表达增加，随后体外从睾丸间质细胞中分泌的 NO 增加。cGMP 特异性磷酸二酯酶 Pde5、Pde6 和 Pde9 上调，而 PRKG1 蛋白在 2 周的睾酮处理后减少。雄激素受体拮抗剂阻断了睾酮诱导的 Leydig 细胞 Nos2 和 Pde5 的诱导。在实验性促性腺激素低下性性腺功能减退症中，NOS2 的表达明显降低，而用睾酮治疗可使 NOS2 的表达增加至对照水平以上。PDE5 蛋白水平在性腺功能减退大鼠中不变，而用睾酮治疗可显著增加其水平。相反，性腺功能减退和睾酮替代减少了 Leydig 细胞中的 PRKG1 蛋白。体外用睾酮处理可导致纯化的 Leydig 细胞中 Nos2 基因表达逐渐增加，随后亚硝酸盐和 cGMP 生成增加。总之，睾酮通过增加 NOS2 表达来上调 NO 信号，并导致 Leydig 细胞中 cGMP 信号的下调。因此，睾酮诱导的 NO-cGMP 信号的调节可能是睾丸类固醇生成的一种有效的自分泌调节剂。
