人精子膜表面电压依赖性阴离子通道的初步研究

目的:研究人类精子膜表面是否存在电压依赖性阴离子通道(VDAC),并研究其生物学特性。方法:设计VDAC的特异性引物,从睾丸cDNA文库中用PCR技术扩增该通道的基因产物,用分子克隆技术体外表达重组VDAC的蛋白质。从精子表面用1%TritonX-100提取及氯仿/甲醇分离疏水性的膜表面蛋白,用免疫印迹法检测精子表面是否存在天然VDAC蛋白质。结果:人类睾丸cDNA文库含有VDAC的基因表达,人类精子膜表面发现靠N端α螺旋连接在精子膜上的VDAC蛋白质。结论:人类精子膜表面镶嵌VDAC蛋白质,该通道是以α螺旋连接在精子膜上,调控精子膜内外离子的转运和信号的转导,对精子的运动和功能至关重要。     

溶脲脲原体感染对精卵结合相关分子硫酸半乳糖甘油酯的影响

目的 :研究溶脲脲原体 (Uu)感染对精卵结合相关分子硫酸半乳糖甘油酯 (SGG)的影响。　方法 :取成熟小鼠附睾精子制成精子悬液 ,随机分为 4组 :Uu组 (与Uu悬液共孵育 )、培养液组 (与Uu培养液共孵育 )、正常组和免疫前血清 (PRS)组。间接免疫荧光法检测SGG在精子膜表面的定位以及Uu对精子膜表面SGG的影响。　结果 :SGG主要位于附睾精子头部相当于顶体区的质膜上。与Uu培养液共孵育的精子 ,其表面SGG的阳性率达82 .0 % ;而与Uu悬液共孵育的精子 ,其表面SGG的阳性率下降至 39.0 % (P =0 .0 0 1)。　结论 :Uu能与精子膜SGG结合 ,SGG可能是Uu在哺乳动物精子膜上的受体 ,Uu封闭精卵结合相关分子SGG可能是Uu感染引起男性不育的分子机制之一。     

正常精子体外与活性氧作用后超微结构观察

目的 :观察精子与活性氧 (ROS)作用后超微结构变化。　方法 :采用Percoll梯度离心法选择具有正常生理功能的精子作为正常精子模型 ,应用次黄嘌呤 黄嘌呤氧化酶体系产生ROS ;在有氧环境下ROS与精子模型孵育后 ,采用透射电镜观察精子超微结构。　结果 :正常精子模型与ROS作用后 ,精子膜及顶体存在不同程度的损伤 ,精子线粒体结构出现异常。　结论 :过多的ROS可致精子膜、顶体以及线粒体的超微结构改变 ,损害精子功能。     

附睾精子蛋白P34H与男性生殖

哺乳动物精子在附睾转运过程中会获得精 卵相互作用所必需的精子表面蛋白。P34H是由人附睾上皮细胞分泌并定位于精子顶体的精子膜蛋白 ,属于短链脱氢酶 还原酶 (SDR)超家族成员。初步研究表明 ,P34H能够介导精子 卵透明带的结合 ,可作为附睾精子成熟的标志物 ,且低水平的附睾精子蛋白P34H与原发性男性不育显著相关。因此 ,精子表面P34H的水平可以作为男性不育症的一个辅助诊断指标。本文对附睾精子蛋白P34H的分子生物学特性、基因的表达与调控、作用机理及其与男性生殖的关系作一综述     

微波辐射对雷达操作人员精子头部及尾部超微结构的影响

目的 研究微波辐射对精子超微结构的影响.方法 利用透射电镜对10例从事微波辐射工作2年以上男性新鲜精液样本中的精子头部及尾部超微结构进行观察.结果 在电镜下微波辐射后男性精子存在多种形态超微结构异常,有以下几种类型:(1)头部异常精子,包括尖头精子、头部含空泡精子.(2)顶体异常精子,包括顶体发育不良、缺失,顶体内形成包涵体精子.(3)尾部异常精子包括尾部形态异常精子(无尾精子、短尾精子)和尾部结构异常精子(线粒体缺失精子、尾部线粒体多种形态和结构异常).结论 微波辐射可导致男性精子项体、头部、尾部线粒体结构异常.     

不育症患者精子头部及尾部超微结构的研究

目的研究不育男性精子超微结构的形态特征。方法利用透射电镜对8例不育男性新鲜精液标本中的精子头及尾部超微结构进行观察。结果在电镜下不育男性精子存在多种形态超微结构异常,有以下几种类型：(1)顶体异常精子,包括顶体膜受损,顶体发育不良、缺失,顶体内形成包涵体精子。(2)头部异常精子,包括尖头精子、圆头精子、头部含空泡精子。(3)尾部异常精子,①尾部形态异常精子,包括无尾精子、短尾精子、卷尾精子、体尾胞质残余。②尾部结构异常精子,包括线粒体缺失精子、尾部线粒体多种形态和结构异常。结论不育男性精子存在顶体、头部、尾部线粒体、微管多种形态和结构异常。     

男性学基础专题的小结

本次大会共收到有关基础理论方面的论文,包括大会交流、书面交流及列题共64篇,其中纯基础理论33篇,基础与临床结合31篇,包括下列几个方面: 一、精子的研究篇数较多,覆盖面较广,计有:1.精子表面唾液酸的测定。比较65例生育、125例不育及妻子流产者64例的精子表面唾液酸与精浆唾液酸的变化,发现精子表面的唾液酸在妻子流产组降低,而精浆内唾液酸无变化;2.用瑞姬氏染色法观察顶体完整性;3.用赖氨酸染色法研究精子核蛋白的变化,发现配偶流产者,核蛋白中赖氨酸增高;4.用精子尾部低渗膨胀试验以测定精子膜功能:(1)从110例原因不明不育病人测定结果发现与精子活     

金黄地鼠腹侧前列腺来源蛋白结合精子表面的实验研究

目的:探讨前列腺分泌蛋白是否可结合于精子表面。方法:以金黄地鼠作为研究对象,应用间接免疫荧光和亲和素标记的蛋白转印方法检测前列腺分泌蛋白是否可结合于精子。制备的抗前列腺粗提取物多克隆抗体,间接免疫荧光技术检测体外与前列腺分泌物孵育的附睾精子,以及体内分别与含前列腺及去除前列腺雄鼠交配后收集的子宫腔内和输卵管腔内精子的前列腺成分抗原。前列腺提取物经电泳分离转膜后和生物素标记附睾精子膜蛋白作用,测定在体外前列腺分泌蛋白能否与附睾精子膜结合。实验分为对照组、附属性腺全去组、腹侧前列腺组、去除腹侧前列腺组。结果:前列腺抗原成分的免疫反应局限在精子体中部表面,而精子头、颈部未见阳性反应。在体外(80±5)%附睾精子与前列腺分泌蛋白结合,在体内与对照组雄鼠交配后收集的子宫腔内精子与前列腺分泌蛋白结合精子阳性率为(30.0±4.6)%,与去除腹侧前列腺组(3.6±1.4)%比较差异有显著性(P<0.01),在体内与对照组雄鼠交配后收集的输卵管腔内精子与前列腺分泌蛋白结合精子阳性率为(16.0±3.6)%,与去除腹侧前列腺组精子(3.2±1.4)%比较差异有显著性(P<0.01)。前列腺分泌蛋白的电泳印记膜与生物素标记附睾精子膜蛋白孵育后显色分析结果可见5条阳性反应带。结论:金黄地鼠的前列腺分泌蛋白可结合于精子体中部表面,前列腺分泌蛋白中至少有5个组分可与精子膜蛋白结合。     

顶体蛋白酶在男性不育症中的应用

顶体蛋白酶是位于精子头部顶体内层浆膜上的一种蛋白溶解酶.已知85%～90%的顶体蛋白酶是以酶原的形式存在.随着精子在体外获能,酶原即可活化.活化的顶体酶一方面溶解顶体膜基质;另一方面又可作用于主要靶器官卵细胞放射冠与透明带,使精子穿入卵细胞,形成受精卵.顶体蛋白酶在受精过程中起着十分重要的作用.因此,男性不育患者与精子顶体蛋白酶活性的低下有密切的关系.本文通过近十余年来,国内有关顶体蛋白酶与男性不育的临床和实验研究作如下综述.     

精子分离两种方法的比较和评价

目的 :探讨王氏管法和精子上游法在精子分离中的应用 ,比较两者的优缺点。　方法 :随机选择 12 7份精液 ,采用 2种方法进行配对处理 ,比较 2种方法分离前后精子活动率、形态、活力和精子顶体形态、精子染色质以及精子膜完整性等参数的变化。　结果 :2种方法处理后的精子活动率、精子活力以及精子形态有显著的差异 (P <0 0 1) ,王氏管法处理后精子顶体完整率、正常染色质率及精子膜完整率亦显示出显著的差异 (P <0 0 1)。　结论 :王氏管法和精子上游法在精子处理中均可获得较好的精子质量 ,但王氏管法则获得更优质的精子 ,且具有方便操作和实用的特点。     

Localization of clusterin on freeze-preserved bull spermatozoa before and after glass wool-sephadex filtration.

Clusterin is a major protein in bull reproductive tract secretions and sperm membrane extract. A polyclonal antibody was produced against clusterin from bull cauda epididymal fluid (CEF) and used for the localization of the protein on bull spermatozoa. Immunoblotting of unreduced bovine samples showed that the anticlusterin antibody reacted with a protein of approximately 94- to 100-kd in rete testis fluid (RTF), a approximately 57- to 76-kd protein in CEF, and with a approximately 57- to 60-kd protein from cauda epididymal sperm membrane extract. The antibody also reacted with stallion RTF and both ram CEF and RTF at relative molecular weights (Mr) that were consistent with the anticipated size of clusterin in these species. Less intense immunostaining was observed for a protein of about 2 times the predicted size of clusterin in unreduced ovine RTF, suggesting the presence of multimers of clusterin in ovine RTF. Also, a dimeric clusterin-sized protein was detected in reduced bovine CEF, suggesting the presence of unprocessed clusterin in bovine CEF. By immunofluorescence, clusterin was detected on only a small fraction of bull spermatozoa, which were morphologically abnormal. Neither permeabilization nor the method of dilution affected the reactivity of the antibody with spermatozoa (P > .05). Average clusterin-positive spermatozoa (CPS) in unpermeabilized, permeabilized, abruptly diluted, and gradually diluted semen were 10.1%, 11.3%, 15.0%, and 14.4%, respectively. CPS were eliminated from semen after filtration through glass wool-Sephadex (GWS) columns. Average CPS in unfiltered and filtered semen were 14.3% and 1.1%, respectively. We conclude that sperm clusterin in bull semen is associated with morphologically abnormal spermatozoa and that clusterin is implicated in the process of abnormal spermatozoa trapping in GWS columns. We suggest that the fraction of CPS in bull semen is a potential marker for poor semen quality.     

Clusterin is up-regulated in glomerular mesangial cells in complement-mediated injury

BACKGROUND: Clusterin is a soluble complement regulatory protein that binds to C5b-7 and inhibits generation of membrane attack complex, C5b-9. Glomerular deposition of clusterin has been observed in human and experimental membranous nephropathy in association with C5b-9 and immune deposits. However, it is controversial as to whether clusterin observed in glomeruli is synthesized by the resident glomerular cells or is derived from the circulation. We examined whether clusterin is expressed by resident glomerular cells exposed to complement-mediated injury. METHODS: In vitro, cultured mesangial cells were exposed to antithymocyte serum immunoglobulin G and 5% normal rat serum as a complement source. In vivo, we induced anti-Thy1 nephritis in rats and examined the kidneys on days 8 and 29. RESULTS: We observed increased expression of clusterin in cultured rat glomerular mesangial cells stimulated by sublytic complement attack. We also demonstrated that in comparison with control rats, both a marked increase in clusterin mRNA in the glomeruli and marked deposition of clusterin protein in the mesangial area occurred in the OX-7-treated rats on day 8 in association with C5b-9 deposition and on day 29. CONCLUSION: Clusterin was induced in glomerular mesangial cells during the course of immune-mediated injuries. This up-regulation of clusterin may play a critical role in protecting mesangial cells from complement attack.     

Clusterin production in the obstructed rabbit kidney: correlations with loss of renal function.

Clusterin, a protein associated with cell death, has been suggested as a marker of renal injury. Correlation of clusterin gene expression with changes in renal function and quantitative measurement of clusterin protein levels after ureteral obstruction have not been previously reported. With unilateral ureteral obstruction in rabbits as the experimental model, the time course of alterations in renal function, clusterin mRNA accumulation, and concentrations of clusterin protein in serum, urine, and renal tissue were investigated. RBF, GFR, and renal concentrating ability (percent sodium reabsorption and urine osmolarity) all decreased (P < 0.05) in the obstructed kidney from control values within 1 day of ureteral obstruction. Clusterin mRNA levels started to rise in the ipsilateral kidney within 12 h of ureteral obstruction and increased up to 10-fold above control levels after 3 days of obstruction. Hybridization histochemistry showed that clusterin mRNA was initially detectable in collecting ducts and distal tubules within 12 h of ureteral obstruction. After 7 days of obstruction, increased accumulation of clusterin mRNA was also detectable in proximal tubular epithelial cells. Clusterin gene expression remained elevated in collecting ducts after 60 days of obstruction. Clusterin expression in the contralateral kidney was increased twofold over control values after 12 h of obstruction. No increase in clusterin mRNA accumulation was detectable after 24 h in the contralateral kidney. Total clusterin protein in the obstructed kidney increased from 0.59 +/- 0.66 (mean +/- 1 SD) to 2.5 +/- 1.3 micrograms after 7 days of ureteral obstruction (P < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)     

Clusterin expression in adult human normal and osteoarthritic articular cartilage

OBJECTIVE: To characterize the expression pattern of clusterin in adult human normal and osteoarthritic cartilage. METHODS: Clusterin mRNA expression in adult human normal and osteoarthritic cartilage was investigated by analysis of cDNA libraries, TaqMan quantitative RT-PCR, microarray and in situ hybridization. RESULTS: Sequence analysis of ESTs from adult human normal and osteoarthritic cartilage cDNA libraries demonstrated that the abundance of clusterin in these libraries was equivalent to genes which have been more commonly associated with cartilage. To examine tissue distribution, TaqMan Quantitative PCR analysis was performed using RNA from a panel of individual normal tissues. Clusterin was expressed at significant levels in cartilage, brain, liver, and pancreas. The expression of clusterin mRNA was up-regulated in early osteoarthritic vs normal cartilage when analysed by microarray analysis. Using in situ hybridization, chondrocytes of normal cartilage expressed moderate levels of clusterin. Upper mid-zone chondrocytes in cartilage with early stages of osteoarthritic disease expressed high levels of clusterin mRNA. In advanced osteoarthritic cartilage, the overall expression of clusterin was reduced. CONCLUSION: The induction of clusterin has been associated with a variety of disease states where it appears to provide a cytoprotective effect. The increased expression of clusterin mRNA in the early stages of osteoarthritis (OA) may reflect an attempt by the chondrocytes to protect and repair the tissue. In contrast, the decrease in clusterin mRNA in the advanced osteoarthritic cartilage accompanies the final degenerative stages of the disease. An understanding of the expression of clusterin in osteoarthritis may allow consideration of this protein as a marker for cartilage changes in this chronic degenerative condition.     

Intrarenal distribution of clusterin following reduction of renal mass.

Clusterin is a multifunctional protein isolated from a number of tissues in several different species. In a variety of renal diseases, clusterin appears in the glomerulus and tubules in association with the membrane attack complex of complement. It is also transiently expressed after several forms of acute renal injury. In this study, we examined the expression and intrarenal distribution of clusterin following subtotal renal ablation. Male rats were subjected to either 1-1/3 nephrectomy (1-1/3 NX), uninephrectomy (UNX) or sham operation (SHAM). Two weeks after surgery, clusterin mRNA was elevated in the 1-1/3 NX group (1-1/3 NX: 1215 +/- 88; UNX: 208 +/- 11; SHAM: 207 +/- 19 OD units; P less than 0.001). Clusterin mRNA increased between 3 and 24 hours after 1-1/3 NX, plateaued, and remained elevated for at least seven weeks. The increased clusterin mRNA in 1-1/3 NX was localized to the tissue adjacent to the infarctive scar (scar 858 +/- 173 vs. non-scar 98 +/- 27 OD units; P less than 0.001). Clusterin protein followed a similar pattern of localization, being increased in most tubules and some peritubular capillaries in the peri-infarct zone. Only occasional tubules were positive for clusterin in the renal tissue distant from the scar or in the kidneys of sham operated rats. Co-localization of clusterin and C5b-9 was not detected. Evidence for apoptosis was found in the peri-infarct zone but not elsewhere in 1-1/3 NX kidney or in the normal kidney following sham operation. Infarction of 1/3 of the left kidney without contralateral nephrectomy, a maneuver which eliminates the compensatory growth, and uremia seen with 1-1/3 NX still resulted in increased clusterin mRNA in the infarcted left kidney compared to the intact right kidney (LK: 790 +/- 112 vs. RK: 128 +/- 25 OD units; P less than 0.001), although the amount of clusterin mRNA was less than that found following 1-1/3 NX. In conclusion, persistently increased clusterin mRNA and protein was seen in the peri-infarct zone following 1-1/3 NX. This increased expression of clusterin may be playing a role in the ischemia-related apoptosis present in the scar-adjacent tissue.     

Glomerular clusterin is associated with PKC-alpha/beta regulation and good outcome of membranous glomerulonephritis in humans

Mechanisms for human membranous glomerulonephritis (MGN) remain elusive. Most up-to-date concepts still rely on the rat model of Passive Heymann Nephritis that derives from an autoimmune response to glomerular megalin, with complement activation and membrane attack complex assembly. Clusterin has been reported as a megalin ligand in immunodeposits, although its role has not been clarified. We studied renal biopsies of 60 MGN patients by immunohistochemistry utilizing antibodies against clusterin, C5b-9, and phosphorylated-protien kinase C (PKC) isoforms (pPKC). In vitro experiments were performed to investigate the role of clusterin during podocyte damage by MGN serum and define clusterin binding to human podocytes, where megalin is known to be absent. Clusterin, C5b-9, and pPKC-alpha/beta showed highly variable glomerular staining, where high clusterin profiles were inversely correlated to C5b-9 and PKC-alpha/beta expression (P=0.029), and co-localized with the low-density lipoprotein receptor (LDL-R). Glomerular clusterin emerged as the single factor influencing proteinuria at multivariate analysis and was associated with a reduction of proteinuria after a follow-up of 1.5 years (-88.1%, P=0.027). Incubation of podocytes with MGN sera determined strong upregulation of pPKC-alpha/beta that was reverted by pre-incubation with clusterin, serum de-complementation, or protein-A treatment. Preliminary in vitro experiments showed podocyte binding of biotinilated clusterin, co-localization with LDL-R and specific binding inhibition with anti-LDL-R antibodies and with specific ligands. These data suggest a central role for glomerular clusterin in MGN as a modulator of inflammation that potentially influences the clinical outcome. Binding of clusterin to the LDL-R might offer an interpretative key for the pathogenesis of MGN in humans.     

Interaction of complement and clusterin in renal injury.

Clusterin is a heterodimeric glycoprotein that has been associated with such diverse biologic functions as reproduction, cell regression, cell aggregation, and regulation of the cytolytic activity of the membrane attack complex of complement. Clusterin is a component of glomerular immune deposits in the kidney, and increased clusterin expression occurs in a number of renal injury states. To further explore the interaction between clusterin and complement, the requirement for an intact complement system for renal clusterin induction in an acute (folic acid nephropathy) and a chronic (subtotal renal ablation) model of renal injury was examined. After it was first demonstrated that folic acid increased renal clusterin mRNA in the rat, a species in which renal clusterin was highly inducible by other stimuli, the effects of folic acid (250 mg/kg ip) on clusterin mRNA and immunoreactivity were examined in mice sufficient and deficient for the fifth component of complement. Similar increases in clusterin mRNA and immunoreactivity were seen in both the C5-sufficient and C5-deficient mice compared with their respective vehicle-injected control groups. Renal clusterin mRNA was also increased to a similar extent in the remaining kidney of both C5-sufficient and C5-deficient mice 10 days after subtotal nephrectomy. In conclusion, the induction of clusterin after folic acid administration or subtotal nephrectomy was independent of the presence of an intact complement system, because similar increases in clusterin expression were observed in C5-sufficient and C5-deficient mice.     

簇蛋白在前列腺上皮细胞凋亡中的表达和作用

目的探讨簇蛋白在前列腺上皮细胞凋亡过程中的表达和作用，及与转化生长因子β1（transforming growth factor β1，TGF-β1）的关系。方法取10只实验犬，分别于去势前及去势后近期（〈15d）和去势后远期（〉15d）取前列腺组织制成石蜡切片，通过免疫组织化学检测前列腺组织中簇蛋白的表达及其表达部位，同时检测TGF-β1的表达。结果去势后近期（〈15d）与去势前的簇蛋白表达有显著性的差异，去势后远期（〉15d）与去势前簇蛋白的表达没有显著性差异；TGF-β1的表达也是如此。结论簇蛋白的表达与前列腺上皮细胞凋亡有关，和TGF-β1表达存在一定的相关性。