摘要目的：研究核糖核酸酶9（RNase9）在男性生殖系统中的表达、定位及功能，为其异常所引起的不育症诊断和治疗提供理论基础。方法采用生物信息学分析人RNase9的结构，预测其功能；利用基因重组技术构建了原核表达载体制备重组RNase9蛋白。通过RT-PCR检测RNase9蛋白在不同组织中的表达，比较老年人（76.3±8.1岁）、成年人（31.2±5.6岁）和胎儿（0.8±0.2岁）附睾组织RNase9蛋白表达量的差异。应用免疫荧光实验观察RNase9蛋白在睾丸、附睾、精子上的定位。应用酵母tRNA底物实验检测RNase9核糖核酸酶活性。分别向精子悬液中加入免疫前血清（A组）和鼠抗RNase9多抗血清（B组），应用抗体封闭精子运动实验检测RNase9蛋白对精子运动能力[平均曲线运动速度（VCL）、平均直线运动速度（VSL）、平均路径速度（VAP）]的影响。8~12周龄金黄地鼠肌注孕激素和人绒毛膜促性腺激素，取卵子与两组精子孵育后（A组精子用免疫前血清，B组精子用RNase9抗血清封闭5 h；实验中所用卵子的数目分别是A组60个和B组62个），应用抗体封闭精子穿卵实验检测RNase9蛋白对精子穿卵能力的影响。结果经过生物信息学分析，RNase9基因属于核糖核酸酶A超家族一新成员，N末端含23个氨基酸的信号肽序列，相对分子质量约为20000，等电点PI 5.92；含5个N?糖基化位点、2个酪蛋白激酶Ⅱ磷酸化位点、1个酪氨酸激酶磷酸化位点、1个NDUFC2位点和2个胰?核糖核酸酶功能域标签位点。RNase9蛋白在人附睾、睾丸、心脏，肺脏、肝脏、脾脏、肾脏、胃多种组织广泛表达，在附睾组织中表达量较高；在成年人附睾组织中的表达量明显高于胎儿和老年人附睾组织（均P<0.05）。RNase9蛋白特异结合在精子的赤道后颈部，原核表达获得高纯度具有生物活性的重组蛋白和相应的抗体。核糖核酸酶实验显示RNase9蛋白缺乏核糖核酸酶活性，为0.111±0.007。抗体封闭精子后不影响精子的运动及穿卵能力，两组VAP、VSL、VCL和受精率差异没有统计学意义（均P>0.05）。结论 RNase9蛋白在附睾及精子上的表达及定位表明该蛋白可能在男性精子成熟及发育过程中起重要作用，但不影响成熟精子的运动及穿卵能力。

abstractsObjective To investigate the expression,localization and function of Ribonuclease 9 (RNase9) in the male reproductive system,so as to provide a theoretical basis for the diagnosis and treatment of infertility caused by abnormal RNase9 expression. Methods Bioinformatic techniques were used to analyze the structure and thereby predict the function of human RNase9. Recombinant human RNase9 protein was obtained using a prokaryotic expression vector constructed by recombinant DNA technology. RT?PCR was performed to evaluate the difference in RNase9 protein expression in various tissues. The RNase9 protein expression in epididymis was compared among elderly[aged(76.3±8.1)years],adults[aged(31.2± 5.6)years]and fetuses [(0.8±0.2)years]. Fluorescence immunohistochemistry was used to localize RNase9 proteins in the testis,epididymis and sperm. Ribonuclease activity of RNase9 was measured with yeast tRNA&nbsp;substrate assay. Sperm suspensions were added with pre?immune serum(Group A)or mouse anti?RNase9 antiserum(Group B),and then subjected to antibody?blocked sperm motility assay to determine the effect of RNase9 protein on indicators of sperm motility[mean curvilinear velocity(VCL),mean straight line velocity (VSL),mean path velocity(VAP)]. From 8 to 12 week?old Golden hamsters intramuscularly injected with progesterone and human chorionic gonadotropin,ova were harvested and incubated with the sperms from Groups A and B(the sperms in Group A had been blocked with pre?immune serum and those in Group B with anti?RNase9 antiserum for 5 h;there were 60 ova used in group A and 62 in Group B). The effect of RNase9 on ovum penetration ability of the anti?body blocked sperms was evaluated by hamster ovum penetration assay. Results Bioinformatic analysis indicated that RNase9 gene is a new member of RNase A superfamily,with a 23?aminoacid signal peptide sequence at the N?terminal,a relative molecular weight of about 20 000 and an isoelectric point(PI)of 5. 92. RNase9 was shown to contain five N?glycosylation sites, two casein kinaseⅡphosphorylation sites,one tyrosine kinase phosphorylation site,one NDUFC2 site and two labeling sites of pancreatic?ribonuclease functional domain. RNase9 protein was ubiquitously expressed in human epididymis,testis,heart,lung,liver,spleen,kidney,and stomach tissues,whereas the expression level was higher in the epididymis. Expression of RNase9 in epididymis was significantly higher in adults than that in the epididymis of fetuses and elderly(all P<0.05). RNase9 protein specifically bound to the equatorial neck region of sperms. Prokaryotic expression yielded to obtain recombinant RNase9 protein and the corresponding antibody with high purity and biological activity. Yeast tRNA substrate assay showed that the RNase9 protein was deficient of ribonuclease activity (0.111 ± 0.007). Antibody blocking was not found to affect the sperm motility of ovum?penetrating ability,with no statistically significant differences in VAP,VSL,VCL and fertilization rate between the two groups(all P>0.05). Conclusion Expression and localization of RNase9 protein in the epididymis and sperm suggest that the protein may play an important role in sperm maturation and development,but may not affect the motility and ovum?penetrating ability of mature sperms rats.