Plac8l1在小鼠精子发生过程中的表达特征研究

Expression characteristics of Plac8l1 in mouse testicular tissue

摘要:

目的：探讨Plac8l1（placenta-specific 8 like 1）基因在小鼠睾丸组织的表达特征，及其与精子发生的相关性。方法：经生物信息学方法初步筛选到一个睾丸特异性表达候选基因Plac8l1，并推测其可能与小鼠精子发生相关；采用半定量RT-PCR和实时定量RT-PCR对Plac8l1表达的时空特异进行了研究；应用原位杂交技术对Plac8l1 mRNA在成年小鼠睾丸中的细胞类群表达特征进行初步研究；通过构建过表达PLAC8L1-EGFP重组蛋白质粒及生物信息学分析进行亚细胞定位研究与功能预测。结果：①Plac8l1是1个睾丸特异性转录基因。②在成年小鼠，Plac8l1只在睾丸和附睾组织发生特异性转录，而且睾丸中的表达水平明显高于附睾[睾丸vs. 附睾=[（1.000±0.000） vs. （0.036±0.018），P=0.000]。③随着小鼠精子发生的起始，睾丸中Plac8l1转录水平逐渐升高（P=0.000）。④mRNA原位杂交结果显示Plac8l1 mRNA主要定位在成年小鼠睾丸中的间质细胞和精母细胞。⑤经生物信息学分析，PLAC8L1也有着一个类似于PLAC8的功能域，过表达重组蛋白显示PLAC8L1在细胞膜上聚集。结论：Plac8l1是1个睾丸特异性基因，可能与精子发生过程中的精母细胞分化相关；PLAC8L1具有1个富含半胱氨酸的PLAC8结构域，这一结构域可能介导了PLAC8L1在细胞膜上聚集并与其他蛋白相互作用，并可能在精母细胞分化中发挥重要作用。

Abstract:

Objective：To study the expression characteristics of Placenta-specific 8 like 1（Plac8l1） in mouse testicular tissue，and to reveal its possible function during spermatogenesis. Methods：The candidate gene Plac8l1 was preliminary screened to be specific ex－pression in testis by bioinformatics methods，and it is speculated that the gene was related to the spermatogenesis. Besides，semi quantitative RT-PCR and real-time quantitative RT-PCR were used to investigate the tissue specificity of plac8l and the temporal and spatial characteristics of the process of spermatogenesis，while in situ hybridization was used to preliminarily study the expression characteristics of Plac8l1 mRNA in the testis of adult mouse. Finally，the subcellular localization was studied and its functional was pre－dicted by constructing PLAC8L1-EGFP recombinant protein particle and bioinformatics analysis. Results：①The results of preliminary bioinformatics showed that Plac8l1 is one of gene for specific transcription. ②Semi-quantitative and quantitative real-time RT-PCR indicated that Plac8l1 only transcript in adult mice testis and epididymis，and testicular expression level was significantly higher than epididymis. ③As the mouse matured，the Plac8l1 transcription level in testis increased gradually. ④RNA in situ hybridization showed that mRNA was mainly positioned in ledig cell and sperm mother cell in mature mouse testis. ⑤Bioinformatics analysis showed that PLAC8L1 also had a function domain similar to the PLAC8 and the over expression of recombinant proteins showed that protein aggregated in the cell membrane. Conclusion：Plac8l1 is a testis specific gene，and is very likely to associate with spermatocyte differ－entiation in the process of spermatogenesis. PLAC8L1 has a cystine which riches in PLAC8 domain，and the domain may mediate PLAC8L1 to aggregate in cell membrane and interact with other protein and play an important role in spermatocyte differentiation.