Ramírez Alfredo R, Castro Maite A, Angulo Constanza, Ramió Laura, Rivera M Montserrat, Torres Mauricio, Rigau Teresa, Rodríguez-Gil Joan E, Concha Ilona I
Instituto de Bioquímica, Facultad de Ciencias, Universidad Austral de Chile, Valdivia, Chile.
Biol Reprod. 2009 Apr;80(4):753-61. doi: 10.1095/biolreprod.108.070961. Epub 2008 Dec 10.
Several studies have shown that dopamine and other catecholamines are present in oviduct luminal fluid. We recently reported that dopamine type 2 receptors (DRD2) are present in a wide range of mammalian sperm, suggesting a role for dopaminergic signaling in events such as fertilization, capacitation, and sperm motility. In the present study, we used Western blot analysis to show that boar sperm express DRD2 and that their activation with dopamine (100 nM) has a positive effect on cell viability that can be correlated with AKT/PKB phosphorylation. Bromocriptine (100 nM) and dopamine (100 nM and 10 muM) increased tyrosine phosphorylation during the capacitation period. Immunofluorescence analysis indicated that DRD2 localization is dynamic and depends on the capacitation stage, colocalizing with tyrosine phosphorylated proteins in the acrosome and midpiece region of capacitated boar sperm. This association was confirmed by coimmunoprecipitation analysis. We also showed that bromocriptine (100 nM) and low-concentration dopamine (100 nM and 10 muM) increased total and progressive motility of sperm. However, high concentrations of dopamine (1 mM) decreased tyrosine phosphorylation and motility in in vitro sperm capacitation assays. This can be explained by the presence of the dopamine transporters (DAT, official symbol SLC6A3) in sperm, as demonstrated by Western blot analysis and immunocytochemistry. Taken together, our results support the idea that dopamine may have a fundamental role during sperm capacitation and motility in situ in the female upper reproductive tract.
多项研究表明,多巴胺和其他儿茶酚胺存在于输卵管管腔液中。我们最近报道,多巴胺2型受体(DRD2)存在于多种哺乳动物精子中,提示多巴胺能信号在受精、获能和精子运动等过程中发挥作用。在本研究中,我们通过蛋白质免疫印迹分析表明,公猪精子表达DRD2,用多巴胺(100 nM)激活DRD2对细胞活力有积极影响,这与AKT/PKB磷酸化相关。溴隐亭(100 nM)和多巴胺(100 nM和10 μM)在获能期间增加酪氨酸磷酸化。免疫荧光分析表明,DRD2的定位是动态的,取决于获能阶段,在获能公猪精子的顶体和中段区域与酪氨酸磷酸化蛋白共定位。这种关联通过免疫共沉淀分析得到证实。我们还表明,溴隐亭(100 nM)和低浓度多巴胺(100 nM和10 μM)增加精子的总活力和渐进性活力。然而,在体外精子获能试验中,高浓度多巴胺(1 mM)降低酪氨酸磷酸化和活力。蛋白质免疫印迹分析和免疫细胞化学表明,这可以通过精子中多巴胺转运体(DAT,官方符号SLC6A3)的存在来解释。综上所述,我们的结果支持多巴胺可能在雌性上生殖道原位精子获能和运动过程中发挥重要作用这一观点。