中华生物医学工程杂志
中華生物醫學工程雜誌
중화생물의학공정잡지
Chinese Journal of Biomedical Engineering
2015年
4期
296-302
,共7页
刘杰%张守信%张成林%孙隽%孙成铭
劉傑%張守信%張成林%孫雋%孫成銘
류걸%장수신%장성림%손준%손성명
RNase9%睾丸%附睾%精子%精子能动性%精子卵子相互作用
RNase9%睪汍%附睪%精子%精子能動性%精子卵子相互作用
RNase9%고환%부고%정자%정자능동성%정자란자상호작용
RNase9%Testis%Epididymis%Sperm%Sperm motility%Sperm-ovum interactions
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。
目的：研究覈糖覈痠酶9（RNase9）在男性生殖繫統中的錶達、定位及功能，為其異常所引起的不育癥診斷和治療提供理論基礎。方法採用生物信息學分析人RNase9的結構，預測其功能；利用基因重組技術構建瞭原覈錶達載體製備重組RNase9蛋白。通過RT-PCR檢測RNase9蛋白在不同組織中的錶達，比較老年人（76.3±8.1歲）、成年人（31.2±5.6歲）和胎兒（0.8±0.2歲）附睪組織RNase9蛋白錶達量的差異。應用免疫熒光實驗觀察RNase9蛋白在睪汍、附睪、精子上的定位。應用酵母tRNA底物實驗檢測RNase9覈糖覈痠酶活性。分彆嚮精子懸液中加入免疫前血清（A組）和鼠抗RNase9多抗血清（B組），應用抗體封閉精子運動實驗檢測RNase9蛋白對精子運動能力[平均麯線運動速度（VCL）、平均直線運動速度（VSL）、平均路徑速度（VAP）]的影響。8~12週齡金黃地鼠肌註孕激素和人絨毛膜促性腺激素，取卵子與兩組精子孵育後（A組精子用免疫前血清，B組精子用RNase9抗血清封閉5 h；實驗中所用卵子的數目分彆是A組60箇和B組62箇），應用抗體封閉精子穿卵實驗檢測RNase9蛋白對精子穿卵能力的影響。結果經過生物信息學分析，RNase9基因屬于覈糖覈痠酶A超傢族一新成員，N末耑含23箇氨基痠的信號肽序列，相對分子質量約為20000，等電點PI 5.92；含5箇N?糖基化位點、2箇酪蛋白激酶Ⅱ燐痠化位點、1箇酪氨痠激酶燐痠化位點、1箇NDUFC2位點和2箇胰?覈糖覈痠酶功能域標籤位點。RNase9蛋白在人附睪、睪汍、心髒，肺髒、肝髒、脾髒、腎髒、胃多種組織廣汎錶達，在附睪組織中錶達量較高；在成年人附睪組織中的錶達量明顯高于胎兒和老年人附睪組織（均P<0.05）。RNase9蛋白特異結閤在精子的赤道後頸部，原覈錶達穫得高純度具有生物活性的重組蛋白和相應的抗體。覈糖覈痠酶實驗顯示RNase9蛋白缺乏覈糖覈痠酶活性，為0.111±0.007。抗體封閉精子後不影響精子的運動及穿卵能力，兩組VAP、VSL、VCL和受精率差異沒有統計學意義（均P>0.05）。結論 RNase9蛋白在附睪及精子上的錶達及定位錶明該蛋白可能在男性精子成熟及髮育過程中起重要作用，但不影響成熟精子的運動及穿卵能力。
목적：연구핵당핵산매9（RNase9）재남성생식계통중적표체、정위급공능，위기이상소인기적불육증진단화치료제공이론기출。방법채용생물신식학분석인RNase9적결구，예측기공능；이용기인중조기술구건료원핵표체재체제비중조RNase9단백。통과RT-PCR검측RNase9단백재불동조직중적표체，비교노년인（76.3±8.1세）、성년인（31.2±5.6세）화태인（0.8±0.2세）부고조직RNase9단백표체량적차이。응용면역형광실험관찰RNase9단백재고환、부고、정자상적정위。응용효모tRNA저물실험검측RNase9핵당핵산매활성。분별향정자현액중가입면역전혈청（A조）화서항RNase9다항혈청（B조），응용항체봉폐정자운동실험검측RNase9단백대정자운동능력[평균곡선운동속도（VCL）、평균직선운동속도（VSL）、평균로경속도（VAP）]적영향。8~12주령금황지서기주잉격소화인융모막촉성선격소，취란자여량조정자부육후（A조정자용면역전혈청，B조정자용RNase9항혈청봉폐5 h；실험중소용란자적수목분별시A조60개화B조62개），응용항체봉폐정자천란실험검측RNase9단백대정자천란능력적영향。결과경과생물신식학분석，RNase9기인속우핵당핵산매A초가족일신성원，N말단함23개안기산적신호태서렬，상대분자질량약위20000，등전점PI 5.92；함5개N?당기화위점、2개락단백격매Ⅱ린산화위점、1개락안산격매린산화위점、1개NDUFC2위점화2개이?핵당핵산매공능역표첨위점。RNase9단백재인부고、고환、심장，폐장、간장、비장、신장、위다충조직엄범표체，재부고조직중표체량교고；재성년인부고조직중적표체량명현고우태인화노년인부고조직（균P<0.05）。RNase9단백특이결합재정자적적도후경부，원핵표체획득고순도구유생물활성적중조단백화상응적항체。핵당핵산매실험현시RNase9단백결핍핵당핵산매활성，위0.111±0.007。항체봉폐정자후불영향정자적운동급천란능력，량조VAP、VSL、VCL화수정솔차이몰유통계학의의（균P>0.05）。결론 RNase9단백재부고급정자상적표체급정위표명해단백가능재남성정자성숙급발육과정중기중요작용，단불영향성숙정자적운동급천란능력。
Objective 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 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.
