目的:探讨D930020B18Rik基因在不同发育阶段小鼠睾丸中的表达规律及其在精子发生中的可能作用。
方法:使用基因表达谱微阵列,我们鉴定了小鼠睾丸中高表达的D930020B18Rik,并通过qPCR分析了该基因的表达模式,免疫组织化学,蛋白质印迹和免疫荧光染色,并利用生物信息学分析验证了其功能和分子机制,双荧光素酶报告基因测定和细胞周期同步。
结果:在出生后的前2周(PNW),D930020B18Rik基因在小鼠睾丸中的表达仍然很低,主要位于精原细胞的细胞质中,从第三次PNW增加到性成熟,位于精原细胞的细胞质和圆形和细长精子细胞的细胞核中,但在成熟精子的细胞核中不存在。系统发育分析表明,D930020B18Rik蛋白序列在哺乳动物中高度保守。基因集富集分析表明,D930020B18Rik及其同源蛋白可能通过参与核质凝聚参与哺乳动物精子发生的调控(归一化富集评分[NES]=1.652,P<0.01,假发现率[FDR]=0.153)。减数分裂(NES=1.960,P<0.01,FDR=0.001)和有丝分裂过程中微管细胞骨架的形成(NES=1.903,P<0.01,FDR=0.009)。双荧光素酶报告基因分析显示,转录因子klf5和foxo1可以识别并结合D930020B18Rik启动子,并执行正或负转录调控功能。
结论:D930020B18Rik基因在小鼠睾丸中以时间和位置特异性的方式表达,与精子生成高度相关,主要位于生殖细胞的细胞核,并可能参与精母细胞减数分裂和精子生成。
OBJECTIVE: To investigate the expression pattern of the D930020B18Rik gene in the testis of the mouse in different stages of development and its possible role in spermatogenesis.
METHODS: Using gene expression profile microarray, we identified highly expressed D930020B18Rik in the mouse testis and analyzed the expression pattern of the gene by qPCR, immunohistochemistry, Western blot and immunofluorescence staining, and verified its function and molecular mechanism using bioinformatics analysis, dual-luciferase reporter assay and cell cycle synchronization.
RESULTS: The expression of the D930020B18Rik gene remained low in the testis of the mouse and mainly localized in the cytoplasm of
spermatogonia during the first 2 postnatal weeks (PNW), increased from the 3rd PNW to sexual maturity, localized in the cytoplasm of
spermatogonia and the nuclei of round and elongated spermatids, but was absent in the nuclei of mature sperm. Phylogenetic analysis showed that the D930020B18Rik protein sequence was highly conserved in mammals. Gene set enrichment analysis indicated that D930020B18Rik and its homologous protein might be involved in regulating spermatogenesis of mammals by participating in nucleoplasmic condensation (normalized enrichment score [NES] = 1.652, P < 0.01, false discovery rate [FDR] = 0.153), meiosis (NES = 1.960, P < 0.01, FDR = 0.001) and formation of microtubule cytoskeleton during mitosis (NES = 1.903, P < 0.01, FDR = 0.009). Dual-luciferase reporter assay revealed that the transcription factors klf5 and foxo1 could identify and bind D930020B18Rik promoters and perform the function of positive or negative transcriptional regulation.
CONCLUSIONS: The D930020B18Rik gene is expressed in the mouse testis in a time- and location-specific manner, highly associated with spermiogenesis, mainly localized in the nuclei of germ cells, and may be involved in the meiosis of spermatocytes and spermiogenesis.