健康人成熟精子中mRNA的种类特征

目的分析具有正常生育能力男性成熟精子mRNA的种类特征。方法健康并育有后代的成年男性11例,禁欲3～5d后取精液,上游法纯化精子,Trizol试剂提取精子总RNA。混合所有总RNA进行基因表达系列分析(SAGE),随后对所得SAGE文库进行功能聚类性分析。结果所建SAGE文库共获877个克隆,测序得到21052个标签,出现两次以上的独特性标签有2712种。SAGE文库用DAVID软件进行功能性聚类分析,高丰度的389个基因能聚合成25组功能性的基因。最突出的是一组核蛋白基因、参与DNA依赖的基因转录和转录调节;其次是与胞质,核糖体蛋白以及蛋白质合成相关的基因;其他基因类别还包括蛋白多肽水解相关蛋白;具有蛋白激酶活性,蛋白氨基酸磷酸化、蛋白修饰相关蛋白;免疫相关蛋白等。结论人成熟精子中存在数量庞大、种类繁多的mRNA,这些mRNA可能并不仅仅是在精子形成过程中残留下来的,它的功能可能包括重新翻译以替代降解的蛋白、染色质的重组装,受精过程中传递一些父系必须的RNAs到卵子中以进行表观遗传。     

利用基因表达连续分析技术构建肝癌HepG2细胞文库

目的 采用基因表达连续分析技术(serial analysis of gene expression,SAGE)建立肝癌HepG2细胞标签文库.方法 提取肝癌HepG2细胞总RNA,分离纯化得到mRNA,利用改进后的M-MLV RTasecDNA合成试剂盒合成生物素标记的双链cDNA;得到全长cDNA后,根据SAGE技术流程后续步骤,建立肝癌HepG2细胞标签文库.结合GenBank、UniGene文库分析标签代表的转录本,最终通过SAGE软件分析标签丰度.结果 15 586个标签中有14 181个获得稳定有效的扩增,长度在200～3000 bp.将获得的长片段进行序列分析后,2023个特异基因被发现.结论 用改进后的逆转录试剂盒可以较好地获得全长cDNA,为SAGE技术构建文库提供可靠的原料.借助SAGE技术建立的肝癌HepG2细胞标签文库,容量大、效率高,为后续基因研究搭建了良好的平台.     

人类精子RNA生殖功能研究进展

人类成熟精子中RNA的存在已经被证实,主要包括mRNA和部分小RNA家族成员。随着研究深入,发现精子mRNA表达差异,与精子活力和男性生育功能相关。而部分精子中特异存在的mRNA和小RNA在精卵融合和早期胚胎发育调控中有重要作用。已经获得的结果提示精源性RNA的表达具有个体差异性,并且是胚胎发育所必需的。对精子RNA功能的深入研究将会对男性不育、人类辅助生育技术以及核移植技术等相关领域产生推动作用。     

应用基因芯片技术研究成年男性精子的基因表达

目的:初步研究健康成人精子中基因的表达情况。方法:收集成年男性活力正常的精子,提取总RNA,以健康成人淋巴细胞总RNA作对照。两组RNA逆转录为cDNA之后,cDNA经酶切、加接头后进行RD扩增,在扩增同时分别掺入Cy3(精子)和Cy5(淋巴细胞)荧光染料标记,标记样品与自制的精子基因表达谱芯片杂交。结果:检测发现在560个基因片段中,有72个基因表达上调,321个基因表达下调,另外167个基因表达无差异。其中与基因复制、转录、翻译及调控等相关的基因表达基本属于无差异表达类型或低表达类型,而与精子发生相关的基因、精子本身的特异性抗原等基因显著高表达,精子中糖酵解相关基因表达上调,而与氧化磷酸化相关的基因则表达下调。结论:在健康成人精子中有丰富的基因表达,而且基因的表达与精子自身的功能和特点相一致。     

弱精子症患者精子中CRISP2基因及蛋白的初步研究

目的:探讨弱精子症患者精子中富含半胱氨酸的分泌蛋白2(CRISP2)mRNA及蛋白的表达水平,探讨其与精子活力的关系及相关的分子机制。方法:收集成年男性弱精子症患者精液78例,活力正常精液70例作为对照组,50% Percoll离心分离纯化后,提取精子总RNA及总蛋白,采用RT-PCR,SYBR Green实时定量PCR和Western印迹技术检测CRISP2 mRNA及蛋白相对表达量。结果:CRISP2基因在弱精子症患者精子中的表达量明显低于其在正常对照组精子中的表达量,下降达4.3倍;Western印迹发现CRISP2蛋白在弱精子症组较正常对照组下降达1.71倍,两组之间的表达量有明显的统计学差异(P<0.05)。结论:CRISP2基因及蛋白在弱精子症患者精子中的表达下调可能与精子活力下降有密切联系,提示CRISP2基因及蛋白可能成为弱精子症发病机制研究的一个分子靶标。     

结肠腺癌及正常黏膜mRNA表达谱的构建

目的构建结肠腺癌和正常黏膜基因的mRNA表达谱,探讨其表达差异,为进一步研究提供线索。方法用SAGE（Serialanalysisofgeneexpression）方法构建两者的mRNA表达文库,挑选一定数量克隆进行测序,结果用SAGE2000软件分析并进行比较。结果构建了同一患者结肠正常黏膜和腺癌的SAGE表达文库,软件分析分别获得标签7926和7672个。癌组织与正常黏膜组织及与国外先期构建的结肠腺癌SAGE文库相比较其高表达基因之间均存在明显的表达差异。结论SAGE技术是构建mRNA表达谱的有效方法;结肠腺癌与正常黏膜高表达基因之间存在明显差别;中国人结肠腺癌的基因表达可能具有自身的特点。     

弱精子症患者精核蛋白表达的检测及意义

目的研究不育症患者精核蛋白mRNA表达量的变化及意义。方法提取6例不育症患者及5例健康男性精子的总RNA,应用RT-PCR法扩增得到精核蛋白cDNA片段;提取精子总碱性核蛋白并进行SDS-聚丙酰胺凝胶电泳,检测其中精核蛋白及组蛋白的含量。结果精子中存在精核蛋白基因的mRNA;不育症患者精核蛋白的含量及精核蛋白mRNA相对含量均下降。结论精核蛋白mRNA相对含量的变化与其蛋白含量的变化一致,可能反映了精子发生过程中某些基因的表达情况。     

TDP-43在男性不育中的研究进展

全世界大约有10%~15%的育龄夫妇婚后1年不孕不育。在不育的原因中,男性因素大约占50%。男性不育病因复杂多样,很多目前尚不能明确。近年来,RNA结合蛋白在男性不育中的作用引起人们的关注。反式激活应答DNA/RNA结合蛋白(TDP-43)是一种DNA/RNA结合蛋白,参与睾丸组织中的基因调控,其异常表达可影响男性生育能力,导致男性不育。TDP-43在正常精子与异常精子中的分布与表达量也是不同的,可能是一种男性不育的标志物。本文主要就TDP-43的结构及其在男性不育中的作用进行综述。     

人精子DNA从头甲基化转移酶及甲基CpG结合蛋白的mRNA表达分析

目的 通过体外分离成熟的精子,利用实时荧光定量PCR(qRealTime-PCR)检测其是否表达DNA从头甲基化转移酶(DNMT1/3a/3b)及甲基CpG结合蛋白(MeCP2),为精子中甲基化表观修饰研究提供依据.方法 体外采集健康男性志愿者的精液标本,利用精子上游收集法获得具有活性的成熟精子.通过SYBR Green/PI双染色法,利用流式细胞术(FCM)检测精子样本的质膜完好性及其活性.抽提精子的总RNA,逆转录PCR获得cDNA,利用qRealTime-PCR鉴定精子甲基化转移酶及甲基CpG结合蛋白的mRNA表达情况.结果 SYBR Green/PI双染色和流式细胞仪检测发现,利用上游收集法可以收集SYBR Green /PI-的精子占总精子数的(94.513±1.120)%,表明该方法可以收集到质膜完好且活性强的精子.qRealTime-PCR结果显示,此样本精子中DNA从头甲基化转移酶的表达比较明显,而甲基CpG结合蛋白的表达量相当低.其中DNMT1的mRNA表达量较高(0.272±0.045,P＜0.05),DNMT3a和DNMT3b的表达水平比较弱[(4.930±0.01)E-006,(6.500±1.7)E-005,P＜0.05],而MeCP2几乎不表达[(2.12±0.91)E-006,p＜0.05].结论 利用qRealTime-PCR可以比较快速方便的检测出此次精子样本中DNA从头甲基化转移酶和甲基CpG结合蛋白的mRNA表达水平.     

高效抗逆转录病毒治疗后HIV感染者/AIDS患者精液质量及HIV RNA水平研究

目的:高效抗逆转录病毒治疗(HAART)可以显著延长HIV感染者的寿命并提高其生活质量,越来越多的男性HIV单阳的夫妻希望完成做父母的愿望,本研究通过检测HAART治疗后男性HIV/AIDS患者精液HIV-1 RNA水平与精液指标,评估潜在传播风险和精液质量,为优生优育提供依据。方法:20例有生育要求且接受HAART超过6个月的男性HIV携带者/AIDS患者,用精子动态图像分析系统检测其精子浓度、存活率、总活力、正常形态精子百分率,用COBAS Amplicor检测精液HIV RNA载量及流式细胞仪技术计数血液CD4+T细胞。结果:20例患者年龄25~40(30.7±5.05)岁,接受HAART治疗6(14.24±12.26)个月以上,治疗前基线CD4+细胞226~965 cells/ul(422.38±200.86),检测时CD4+细胞341~1 058 cells/ul(535.76±212.02)。除1例外精液量均高于正常参考值(≥2 ml);14例患者精子总活力低于正常值(≥40%),18例患者前向运动精子百分率低于正常参考值(≥32%),2例精子浓度低于正常参考值(≥15×106/ml);有5例精子存活率低于正常参考值(58%);有6例正常形态精子百分率≤4%。20例患者外周血HIV-1 RNA均20拷贝/ml,18例精浆HIV-1RNA也20拷贝/ml,但2例20拷贝/ml,分别为4.70×101拷贝/ml和2.2×102拷贝/ml。4例与配偶无保护措施同房超过半年,女方定期查HIV抗体均阴性,其中1例自然怀孕生育,母子HIV-1 RNA均为阴性。结论:HAART治疗后仍有部分患者精液中HIV RNA水平高于血浆中HIV RNA水平,提示精液仍可能存在少量HIV,具有潜在传染风险,因此对男性HIV感染者/AIDS患者常规检查精液HIV-1 RNA可以降低女性感染风险。同时发现,HIV感染者/AIDS患者精子总活力与浓度低于正常参考值,生育能力降低。     

Apoptosis and kinematics of ejaculated spermatozoa in patients with varicocele

Increased DNA fragmentation is found in sperm from infertile men. Varicocele is an important cause of male infertility, even though it is present in 15% of men who father children. Semen analysis does not always identify infertility in these patients. Sperm motility is strongly correlated with male fertility potential. The goal of this study was to determine the correlation between apoptosis and kinematics in the ejaculated spermatozoa of patients affected by varicocele. Fresh semen samples were obtained from 30 patients with varicocele and 15 fertile controls. These samples were compared using computer-assisted semen analysis and were assayed to determine the degree of sperm apoptosis. The apoptotic index (AI) was calculated by dividing the number of terminal deoxynucleotidyl transferase-mediated deoxyuridine-5'-triphosphate nick end labeling (TUNEL) stained spermatozoa by the total number of Hoechst 33258-stained sperm cells for 300 sperm. Five microscopic fields were analyzed to obtain 5 AIs for each individual. Results demonstrated no significant difference in semen quality and sperm motion characteristics; however, a significantly higher AI (23.05% +/- 4.07%: mean difference +/- SE, 95% CI, 15.06%-31.03%, P <.0001) was identified in the varicocele group than in the fertile controls. We concluded that sperm apoptosis does not seem to correlate with semen quality and sperm kinematics and that apoptosis is increased in ejaculated spermatozoa in patients with varicocele compared to normal fertile men.     

Spermatozoa protein alterations in infertile men with bilateral varicocele

Among infertile men, a diagnosis of unilateral varicocele is made in 90% of varicocele cases and bilateral in the remaining varicocele cases. However, there are reports of under-diagnosis of bilateral varicocele among infertile men and that its prevalence is greater than 10%. In this prospective study, we aimed to examine the differentially expressed proteins (DEP) extracted from spermatozoa cells of patients with bilateral varicocele and fertile donors. Subjects consisted of 17 men diagnosed with bilateral varicocele and 10 proven fertile men as healthy controls. Using the LTQ-orbitrap elite hybrid mass spectrometry system, proteomic analysis was done on pooled samples from 3 patients with bilateral varicocele and 5 fertile men. From these samples, 73 DEP were identified of which 58 proteins were differentially expressed, with 7 proteins unique to the bilateral varicocele group and 8 proteins to the fertile control group. Majority of the DEPs were observed to be associated with metabolic processes, stress responses, oxidoreductase activity, enzyme regulation, and immune system processes. Seven DEP were involved in sperm function such as capacitation, motility, and sperm-zona binding. Proteins TEKT3 and TCP11 were validated by Western blot analysis and may serve as potential biomarkers for bilateral varicocele. In this study, we have demonstrated for the first time the presence of DEP and identified proteins with distinct reproductive functions which are altered in infertile men with bilateral varicocele. Functional proteomic profiling provides insight into the mechanistic implications of bilateral varicocele-associated male infertility.     

Functional aspects of human sperm binding to the zona pellucida using the hemizona assay

The hemizona assay (HZA) has facilitated investigations of sperm function in relation to zona pellucida binding. In this study, the authors examined: 1) the association between hyperactivated sperm motility and HZA binding; 2) the binding kinetics and efficiency of sperm from subfertile men; and 3) the influence of sperm freezing and thawing on binding capacity. For each HZA, a nonviable human oocyte was cut into equal zona hemispheres. The mean number of bound sperm and the incidence of hyperactivation were significantly greater for samples of sperm from fertile men compared with sperm from subfertile men (P less than 0.05). Subfertile sperm had a binding curve that paralleled the curve for fertile sperm, although the magnitude of binding was markedly reduced. Freezing and thawing of sperm from fertile samples impaired their capacity to bind to the zona pellucida. The HZA binding efficiency was reduced by 30%, although the binding curves for fresh versus frozen samples remained parallel.     

上海地区正常生育力男性精液参考值初探

目的:回顾分析上海地区志愿捐精者与正常生育力男性精液分析各项主要参数的分布特征,比较两组男性精液质量的差别,探讨上海地区男性精液参数的正常参考值下限。方法:2010年10月至2011年7月上海市人类精子库招募正常生育力男性41例,健康捐精者100例,按《世界卫生组织人类精液检查与处理实验室手册》(第5版)进行精液常规检测,评估精液体积、精子浓度、前向运动(PR)精子百分率、精子总数和PR精子总数的均值,标准差,并进行t检验。同时统计正常生育力组上述各参数的分布,得出精液特征参数的正常参考值下限。结果:健康捐精组与正常生育力组精液常规各项主要参数(精液体积、精子浓度、PR精子百分率、精子总数、PR精子总数)间差异无统计学意义(P<0.05)。上海地区正常生育力男性精液参考值下限(P<0.05)为:浓度≥27.3×106/ml、PR≥8.1%、体积≥0.82 ml、精子总数≥44.73×106/1次射精、PR精子总数≥24.68×106/1次射精。结论:在评估男性生育力时,精子总数和PR精子总数可能是比精子浓度、精液体积和PR精子百分数更具参考价值的评价指标。     

Sperm loss in the urine of sexually rested men

In the majority of species, the number of spermatozoa ejaculated is considerably less than the number of sperm produced. Two possible explanations for this discrepancy are the resorption of sperm in the reproductive tract and/or the passage of sperm to the urine. We have examined 24-h urine samples from fertile men who abstained from sexual activity for up to 13 days. Very few sperm were found in the 24-h urine samples. We conclude that sperm loss in the urine is not a significant pathway of sperm disposal in the sexually rested male.     

The cytochemical localization of ATPase activity in the spermatozoa from fertile and infertile human ejaculate by electron microscope (author's transl)]

The cytochemical localization and intensity of adenosine triphosphatase (ATPase) activity in the spermatozoa from fertile and infertile human ejaculate were observed by an electron microscope. Sperm from fertile and infertile human ejaculate were fixed in 1% glutaraldehyde and treated histochemically to demonstrate calcium- and magnesium-dependent ATPase (Ca++- and Mg++-dependent). Furthermore, as substrates, ADP, AMP, and beta-glycerophosphate were used. The localization of Ca++-activated ATPase was not different from that of Mg++-activated ATPase. In the fertile human ejaculated sperm, ATPase activity was found on the surface of the acrosome and mitochondria consisting of the mitochondrial sheath, around the outer coarse fibers and in the axial filament complex. Compared with the result with fertile specimens, in the infertile human ejaculated sperm, ATPase activity on the motile structures, the outer coarse fibers, and the axial filament complex were considerably weaker and occasionally not recognized. From this study, it may be considered that ATPase around the outer coarse fibers and in the axial filament complex of sperm may serve to mediate contraction of the axonemal elements during motility. (Author's Modified)     

Seminal arginase activity in infertility

Arginase (Arg) activity in seminal plasma and sperm cells from infertile men and healthy fertile donors was measured. There were no statistically meaningful differences in seminal plasma Arg activity between the two groups whereas sperm cells from oligospermic infertile men had a higher Arg activity compared with the controls. Some important correlations were established between sperm count and Arg activity (negative values) and sperm motility and Arg activity (positive values) in both sperm cells and plasma samples from infertile men. Results suggest that the arginine-nitric oxide pathway within sperm cells from oligospermic infertile men is disturbed by enhanced Arg activity. We think that this may play a part in sperm dysfunction and male infertility. Received: 22 October 1998 / Accepted: 1 October 1999