小鼠精子发生相关蛋白3基因在小鼠生精细胞的特异表达及对HEK 293T细胞凋亡和自噬的影响

Novel expression of spermatogenesis-associated protein 3 gene in mouse spermatogenic cells and its influence upon apoptosis and autophagy in HEK 293T cells

目的检测小鼠精子发生相关蛋白3(spata3)基因在小鼠生精细胞的表达情况,并借助过表达细胞模型进一步分析该基因对人胚肾HEK 293T细胞凋亡及自噬的影响,旨在探讨spata3在精子发生过程中的意义。方法分别采用RT-PCR和免疫印迹法检测spata3基因mRNA及蛋白产物在小鼠各组织中的表达;应用免疫组织化学和免疫荧光染色观察SPATA3蛋白在生精细胞中的定位;借助脂质体将真核表达载体Plv-EGFP-2(a)purospata3瞬时转染HEK 293T细胞,进一步在蛋白水平分析细胞凋亡相关蛋白Caspase-3、多聚ADP-核糖聚合酶(PARP)、BAX和Bcl-2及自噬相关蛋白LC3A/B的变化。结果 Spata3基因及其编码产物在小鼠睾丸组织特异表达;粗线期精母细胞和圆形精子细胞的胞质与胞核均有显著SPATA3蛋白的阳性着色,长形精子细胞的胞质也有大量分布;过表达spata3的HEK 293T细胞内活化型Caspase-3和PARP降解产物的含量较对照组差异无显著性,BAX表达量0.815±0.020较裸细胞组0.469±0.012和空载体转染组0.588±0.018均有所增高,Bcl-2含量0.214±0.020低于裸细胞0.507±0.021和空载体转染组0.545±0.024,LC3A/B-Ⅱ的表达量0.741±0.037则显著高于裸细胞组0.136±0.011和空载体转染组0.169±0.012。结论 Spata3基因在小鼠生精细胞特异表达,过表达spata3对HEK 293T细胞凋亡无明显影响,但可以促进细胞自噬。

Objective To detect the expression of spermatogenesis-associated protein 3（ spata3） gene in mouse spermatogenic cells and to analyze its probable function on apoptosis and autophagy in HEK 293 T cells over-expressed spata3. The overall aim is to explore the possible significance of spata3 during mouse spermatogenesis. Methods Semiquantitative reverse transcription polymerase chain reaction（ RT-PCR） and Western blotting were used to detect the expression of spata3 mRNA and its encoded protein in a variety of mouse tissues,respectively. Immunohistochemical staining and indirect immunofluorescent staining were used to locate the SPATA3 protein in spermatogenic cells. The recombinant eukaryotic expression plasmid Plv-EGFP-2（ a） puro-spata3 were transiently transfected into HEK 293 T cells using liposome and the expression level of apoptosis-related proteins including cleaved-Caspase3, poly ADP-ribose polymerase（ PARP）,BAX,Bcl-2 and autophagy-related protein LC3 A/B were investigated by Western blotting. Results Spata3 mRNA and its encoded product were specifically expressed in mouse testis. SPATA3 positive signals were mainlylocated in the cytoplasm and the nucleus of both the round spermatids and the pachytene spermatocytes. Moreover,SPATA3 stainning signals were detected in the cytoplasm of elongating spermatids. The expression levels of cleaved-Caspase-3 and cleaved-PARP were not significant difference between HEK 293 T cells over-expressed spata3 and control groups. The content of BAX in 293 T cells over-expressed spata3 0. 815 ± 0. 020 was higher than that in 293 T cells 0. 469 ± 0. 012 and in293 T cells transfected with Plv-EGFP-2（ a） puro 0. 588 ± 0. 018,while the expression of Bcl-2 in HEK 293 T cells overexpressed spata3 0. 214 ± 0. 020 was lower than that in HEK 293 T cells 0. 507 ± 0. 021 and in HEK 293 T cells transfected with Plv-EGFP-2（ a） puro 0. 545 ± 0. 024. The amount of LC3 A/B-Ⅱin HEK 293 T cells over-expressed spata3 0. 741 ±0. 037 were detected higher than that in HEK 293 T cells 0. 136 ± 0. 011 and in HEK 293 T cells transfected with Plv-EGFP-2（ a） puro 0. 169 ± 0. 012. Conclusion Spata3 is specifically expressed in mouse spermatogenic cells and its overexpression in HEK 293 T cells has no significant effect upon apoptosis,but could promote autophagy.