The Wnt/β-catenin pathway participates in many important physiological events such as cell proliferation and differentiation in the male reproductive system. We found that Kinesin-2 motor KIF3A is highly expressed during spermatogenesis in Eriocheir sinensis; it may potentially promote the intracellular transport of cargoes in this process. However, only a few studies have focused on the relationship between KIF3A and the Wnt/β-catenin pathway in the male reproductive system of decapod crustaceans. In this study, we cloned and characterized the CDS of β-catenin in E. sinensis for the first time. Fluorescence in situ hybridization and immunofluorescence results showed the colocalization of Es-KIF3A and Es-β-catenin at the mRNA and the protein level respectively. To further explore the regulatory function of Es-KIF3A to the Wnt/β-catenin pathway, the es-kif3a was knocked down by double-stranded RNA (dsRNA) in vivo and in primary cultured cells in testes of E. sinensis. Results showed that the expression of es-β-catenin and es-dvl were decreased in the es-kif3a knockdown group. The protein expression level of Es-β-catenin was also reduced and the location of Es-β-catenin was changed from nucleus to cytoplasm in the late stage of spermatogenesis when es-kif3a was knocked down. Besides, the co-IP result demonstrated that Es-KIF3A could bind with Es-β-catenin. In summary, this study indicates that Es-KIF3A can positively regulate the Wnt/β-catenin pathway during spermatogenesis and Es-KIF3A can bind with Es-β-catenin to facilitate the nuclear translocation of Es-β-catenin.

译文

Wnt /β-catenin途径参与许多重要的生理事件,例如男性生殖系统中的细胞增殖和分化。我们发现Kinosin-2电机KIF3A在中华绒螯蟹的精子发生过程中高度表达。它可能会在此过程中促进货物的细胞内运输。然而,只有很少的研究集中在十足纲甲壳纲雄性生殖系统中KIF3A与Wnt /β-catenin途径之间的关系上。在这项研究中,我们首次克隆和表征了中华绒螯蟹中β-catenin的CDS。荧光原位杂交和免疫荧光结果显示Es-KIF3A和Es-β-catenin在mRNA和蛋白水平上共定位。为了进一步探索Es-KIF3A对Wnt /β-catenin途径的调控功能,在体内和中华绒螯蟹的原代培养细胞中,通过双链RNA(dsRNA)敲除了es-kif3a。结果表明,在es-kif3a敲除组中,es-β-catenin和es-dvl的表达降低。当es-kif3a被击倒时,精子形成后期Es-β-catenin的蛋白表达水平也降低,Es-β-catenin的位置从细胞核改变为细胞质。另外,co-IP结果表明Es-KIF3A可以与Es-β-catenin结合。总之,这项研究表明,Es-KIF3A可以在精子发生过程中正调控Wnt /β-catenin途径,Es-KIF3A可以与Es-β-catenin结合,促进Es-β-catenin的核转运。

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