C14orf48基因在人精液精子中的表达和生物信息学分析

Expression and bioinformatics analysis of C14orf48 in ejaculated spermatozoa from normozoospermic and asthenozoospermic men

目的筛选与弱精子症发生相关的基因。方法采集正常男性和弱精患者精液精子,非连续梯度离心法分离纯化精子,收集95%percoll以下和57%与76%Percoll层之间的精子,以排除生精细胞和白细胞的污染。将纯化后精子的cDNA探针与Affymetrix全基因组芯片杂交。筛选出差异表达的基因,利用生物信息学方法对该基因进行分析。用RT-PCR验证该基因在正常男性和弱精患者精液精子中的差异表达,并用RT-PCR方法分析该基因在人不同组织(心、肝、脾、肺、肾、胃、脑、睾丸)中的表达。结果分析芯片杂交结果,筛选出一个差异表达杂交点C14orf48(GeneBank登录号BC031252),该基因在正常男性和弱精子症患者精液精子中检测到的信号值均化后分别是141.2(P)和2.8(A)。生物信息学分析显示,该基因为全长1952的完整ORF,编码140个氨基酸、分子量为15.808 kD的功能未知蛋白质Q8NCU1。该基因在人睾丸组织中特异性表达,亚细胞定位预测该基因在细胞质(39.1%)和细胞核(34.8%)中表达。RT-PCR分析结果表明C14orf48在弱精子症患者精液精子中不表达,在正常男性精液精子中强烈表达,在检测人的8种组织中,也只有在睾丸组织中表达。结论 C14orf48基因为睾丸特异性表达基因,其在弱精子症患者精液精子中不表达,提示其表达水平的缺失可能在弱精发中起着重要作用。

Objective To identify genes involved in asthenozoospermia.Methods Semen samples were obtained from normozoospermic and asthenozoospermic volunteers and semen analysis was performed according to WHO criteria.To rule out the contamination of germ cells and leucocytes,human ejaculated spermatozoa were purified by a discontinuous Percoll density gradient centrifugation.CDNA samples from purified sperm of two groups were performed with human whole genome Affymetrix chip analysis to screen the differently expressed genes.The characteristics of the selected gene were analyzed by various bioinformatics tools.RT-PCR was used here to compare the selected gene in ejaculated spermatozoa from normozoospermic and asthenozoospermic men,and to identify the expression profile of the selected genes in human tissues.Results By analysing the hybridization signals,a gene C14orf48（gb：BC031252） with differential expression in the normozoospermic（141.2,P） and asthenozoospermic men（2.8,A） was identified,which function is unknown.It reperents an open reading frame of 1950 bp which encoded a putative protein of 140 amino acids,Q8NCU1.By bioinformatics analysis,C14orf48 gene is testis-specific gene,and located in cytoplasm（39.1%） and nuclear（34.8%）.RTPCR analysis showed that the expression of C14orf48 gene was strong in normozoospermic men,but not in asthenozoospermic men,and showed that C14orf48 gene expressed exclusively in human testis.Conclusion The expression of C14orf48 in ejaculated spermatozoa was significantly decreased in asthenozoospermic men,which is one possible reason for low sperm motility.Well understanding of the molecular function of C14orf48 will be important to the elucidation of the molecular events underlying mammalian male asthenozoospermia.