AKT1激酶在睾丸中的功能及其对LMNA蛋白磷酸化修饰的影响

Functions of AKT1 kinase in testis and its effects on phosphorylation modification of LMNA protein

  • 摘要: 【目的】探究丝氨酸/苏氨酸蛋白激酶1(AKT1)在睾丸精子发生中的功能及其对核纤层蛋白A/C(LMNA)磷酸化修饰的影响,为深入理解雄性生殖调控网络提供理论依据。【方法】采集性成熟期水牛睾丸组织,采用免疫组织化学染色检测AKT1及AKT1(pS473)的分布。建立睾丸组织体外培养模型,分别添加抑制剂A-674563和激活剂SC79干预AKT1活性。利用苏木精—伊红染色观察睾丸组织形态,采用试剂盒检测细胞凋亡率和增殖率,通过Western blotting检测凋亡相关蛋白Bax、Bcl2和Caspase3的相对表达量及LMNA蛋白Ser392位点的磷酸化水平。【结果】AKT1激酶及AKT1(pS473)广泛分布于曲细精管各类型生精细胞中,主要定位于细胞质。随着A-674563浓度升高,睾丸曲细精管结构逐渐松散,生精细胞数量减少,细胞间隙增大。抑制AKT1激酶后,与Control组相比,500 nmol/L A-674563组细胞凋亡率极显著升高(P<0.01,下同),100、500和1000 nmol/L A-674563组细胞增殖率极显著降低;500和1000 nmol/L A-674563组促凋亡蛋白Bax和Casepase3相对表达量极显著升高,500和1000 nmol/L A-674563组抑凋亡蛋白Bcl2的相对表达量极显著降低;500 nmol/L A-674563组LMNA(pS392)蛋白相对表达量显著降低(P<0.05),1000 nmol/L A-674563组LMNA(pS392)蛋白相对表达量极显著降低。激活AKT1激酶后,与Control组相比,SC79组细胞凋亡率极显著降低,细胞增殖率极显著升高;AKT1(pS473)和LMNA(pS392)蛋白相对表达量极显著升高。【结论】AKT1激酶是睾丸细胞存活与增殖的关键调控因子,其通过调节LMNA蛋白Ser392位点磷酸化水平,参与细胞分裂过程的调控。抑制AKT1激酶可降低LMNA蛋白磷酸化水平,破坏睾丸组织形态结构并诱导生精细胞凋亡。激活AKT1激酶可促进LMNA蛋白磷酸化修饰,增强睾丸组织细胞增殖活力。

     

    Abstract: 【Objective】This study aimed to investigate the functions of serine/threonine protein kinase 1 (AKT1) in testicular spermatogenesis and effects on phosphorylation modification of nuclear lamina protein A/C (LMNA), thereby providing theoretical reference for elucidating male reproductive regulatory networks.【Method】Testicular tissues from mature buffaloes were collected for immunohistochemical staining to detect the distribution of AKT1 and AKT1 (pS473). An in vitro culture model of testicular tissues was established, in which the inhibitor A-674563 and activator SC79 were added respectively to intervene AKT1 activity. The morphology of testicular tissues was observed by hematoxylin-eosin staining. The cell apoptosis rate and proliferation rate were determined using assay kits, while the relative expression of apoptosis-related proteins (Bax, Bcl2, and Caspase 3) and phosphorylation level of LMNA protein at Ser392 locus were detected by Western blotting.【Result】AKT1 and AKT1 (pS473) were widely distributed in types of spermatogenic cells at seminiferous tubules, mainly locating at cytoplasm. As the concentration of A-674563 increased, the structure of the seminiferous tubules began to be disorganized, the spermatogenic cell number was reduced, and intercellular spaces were widened. Compared with the control and after inhibition of AKT1 kinase, the 500 nmol/L A-674563 group showed an extremely significant increase in the apoptosis rate (P<0.01, the same below), and the cell proliferation rate under treatments of 100, 500, and 1000 nmol/L A-674563 showed an extremely significant decrease. The relative expression of pro-apoptotic proteins BAX and Caspase 3 in the 500 and 1000 nmol/L A-674563 groups extremely significantly increased, and the relative expression of anti-apoptotic protein Bcl2 in the 500 and 1000 nmol/L A-674563 groups extremely significantly decreased. The relative expression of LMNA (pS392) protein in the 500 nmol/L A-674563 group significantly decreased (P<0.05). The relative expression of LMNA (pS392) protein in the 1000 nmol/L A-674563 group was extremely significantly decreased. Compared with the control and after activating the AKT1 kinase, the cell apoptosis rate of SC79 group extremely significantly decreased, and the cell proliferation rate extremely significantly increased; the relative expression of AKT1 (pS473) and LMNA (pS392) proteins extremely significantly increased.【Conclusion】The AKT1 kinase is the key regulatory factor for survival and proliferation of testicular cells, which regulates the process of cell division by modulating the phosphorylation level of LMNA protein at Ser392 locus. The inhibition of AKT1 kinase can reduce phosphorylation level of LMNA protein, disrupt the morphological structure of testicular tissue structure, and induce apoptosis of spermatogenic cells. The activation of AKT1 kinase can promote phosphorylation modification of LMNA protein and increase proliferation activity of testicular tissue cells.

     

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