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     

附睾蛋白酶抑制剂Eppin的研究进展

附睾蛋白酶抑制剂Eppin是一种睾丸和附睾特异性分泌的蛋白质,精液中含量极为丰富,精子表面大量存在,用重组Eppin免疫的猴子出现不育,Eppin有望成为人类有效的可逆性的免疫避孕疫苗。现就Eppin基因及其蛋白质结构功能特点,Eppin引起免疫性不育的分子机制以及Eppin调控PSA水解精囊蛋白Semenogelin的研究进展进行了综述。     

精液凝固蛋白Semenogelin和附睾蛋白酶抑制剂Eppin的研究进展

精液凝固蛋白Semenogelin (Sg)与附睾蛋白酶抑制剂Eppin参与精液凝胶的形成,并调控精液液化.Sg通过Eppin与精子结合可抑制精子活动.抗Eppin抗体与Sg对精子活动有着类似的抑制作用,重组Eppin免疫的猴子出现不育,从不育猴子提取的抗Eppin抗体在体外可显著抑制人类精子的活动.现就Sg、Eppin在精液凝固过程中的相互作用,抗Eppin抗体、Sg对精子抑制作用及其分子机制的研究进展作一综述.     

附睾环节的节育研究现况

附睾是男性生殖系统的一个重要器官 ,是精子成熟与贮存的场所。现公认最为理想的男用避孕环节是干扰精子成熟以及附睾机能。一些抗生育药物可直接作用于附睾 ,阻止精子正常发育 ;用附睾及精浆等分泌的某些具生物活性的蛋白去免疫动物 ,也能导致动物不育或生育能力下降。这些无疑为开发新型男性避孕、节育方法指明方向。     

附睾环节的节育研究概况

附睾是男性生殖系统的一个重要器官,是精子成熟与贮存的场所。现公认最为理想的男用避孕环节是干扰精子成熟以及附睾机能。一些抗生育药物可直接作用于附睾,阻止精子正常发育;用附睾及精浆等分泌的某些具生物活性的蛋白去免疫动物,也能导致动物不育或生育下降。这些无疑为开发新型男性避孕、节育方法指明方向。     

附睾相关的抗生育研究及进展

虽然至今尚没有一种直接作用于附睾并可供临床试验用的男性避孕药 ,但对附睾功能和精子在附睾成熟的研究表明 ,附睾极有可能成为男性抗生育最理想的靶器官。相关的动物试验包括直接作用于附睾精子如磺胺水杨嗪类、影响能量代谢与精子活动的氯化甘油和氯化糖苷类化合物、影响附睾环境如雷公藤单体和铜粉等。对附睾特异蛋白研究 ,可望发现免疫学途径的靶抗原 ,用于制备避孕疫苗。近年国内外对附睾特异表达基因的研究取得某些进展 ,如SC342、bin1基因的克隆和功能研究 ,有助于临床附睾炎和不育症的诊治 ,同时有望成为干扰附睾精子成熟达到避孕目的的新靶点。     

附睾功能基因的研究方法及进展

精子的成熟不是靠自身来完成,而是通过与附睾管腔中的微环境相互作用而取得。搞清精子成熟的机制和调控,将为新的男性避孕药、诊断和治疗男性不育症、性传播疾病的防治等方面提供一系列新的靶位点。本文分别从基因、蛋白、基因组与蛋白质组三个方面综述了附睾功能基因的研究方法及进展。     

介绍一种测定大鼠附睾精子活率和密度的改进方法

介绍一种测定大鼠附睾精子活率和密度的改进方法林宁，许烨关键词精子活率，精子密度，附睾，棉酚，大鼠中图法分类号Ｒ４４６．１９本实验室多年来从事男性生殖的研究，在男用节育药筛选方面做了大量工作。对大鼠附睾精子的检测是男性生殖研究中一个重要实验手段，我们参...     

附睾氧化应激研究进展

附睾是男性生殖系统的重要器官之一,负责精子的成熟、转运和储存。附睾功能的实现依赖于其旺盛的生理活性,因而自由基的产生不可避免。自由基过度积累势必造成附睾氧化应激,精子质膜及其DNA发生氧化损伤,极大影响精子运动和受精能力的获得,从而造成男性不育。本文对附睾氧化应激在能量代谢、炎症反应等过程中的产生机制和超氧化物歧化酶、过氧化氢酶、谷胱甘肽过氧化酶、吲哚胺双加氧酶、还原性谷胱甘肽及硫氧还蛋白等物质的抗氧化作用进行了综述,以期为男性不育症的有效预防、诊断和治疗提供新思路。     

水通道蛋白抑制剂对大鼠精子成熟和生育影响的研究

目的通过水通道蛋白抑制剂对大鼠附睾精子成熟的影响，探索睾丸后抗生育作用靶点。方法不同剂量乙酰唑胺大鼠灌胃给药，连续10d。结束时将受试雄鼠与雌鼠合笼，计算交配指数、受孕指数、生育指数。采用计算机辅助分析系统分析精子活力形态。使用酶联免疫吸附法测定大鼠血清睾酮（T）和双氢睾酮（DHT）的浓度。睾丸附睾称重后进行HE染色和电镜分析。结果乙酰唑胺各剂量组大鼠精子活力明显降低，同时畸形率显著增加，导致雌鼠受孕率下降，而T、DHT和交配指数均无明显变化，睾丸和附睾亦无明显病理学改变。结论水通道蛋白抑制剂乙酰唑胺可以通过影响大鼠附睾精子成熟来抑制雄鼠生育，同时不影响睾丸和生殖内分泌系统。     

胱蛋白酶抑制剂相关的附睾精子发生基因的调节和功能

CRES(cystatin relatedepididymalspermatogenic,胱蛋白酶抑制剂相关的附睾精子发生 )蛋白是胱蛋白酶抑制剂(cystatin)超家族中家族 2的一个亚类。然而 ,与cystatinC的广泛性表达不同 ,Cres基因只在分裂后的生殖细胞、附睾头部近端和腺垂体促性腺激素细胞中表达。cystatin对C1半胱氨酸蛋白酶抑制作用的发挥必须有 3个共有位点的参与 ,而CRES蛋白缺少其中的 2个位点。因此 ,CERS在生殖系统和神经内分泌系统中的功能也许是独特的和组织特异性的。本文概述了以下方面的研究 :①Cres基因启动子及其转录调节蛋白相关的可能对Cres的组织特异性表达有重要作用的DNA结合位点。②CRES蛋白的生物学功能。Norethern印迹法、凝胶转移分析和瞬时转染实验均表明 ,附睾和促性腺激素细胞中主要表达C EBP家族中的C EBPβ(CCAAT 增强子结合蛋白 ) ,且C EBPβ对于Cres基因在这两个组织中的高表达是必不可少的。另外 ,构建了表达氯霉素乙酰基转移酶 (CAT)报告基因的转基因小鼠 ,CAT报告基因由Cres基因 5′端 1 6kb的启动子所调控。分析显示 ,CATmRNA仅在生殖细胞中表达 ,说明Cres 5′端 1 6kb的侧翼区含有调控CAT在睾丸中表达的DNA序列 ,而缺乏指导CAT在附睾中表达的序列 ,或者此1 6kb的DNA片段存在Cres的负调节成分。最后 ,     

Protease activation during surgical stress in the rat small intestine

BACKGROUND: Surgical stress affects intestinal permeability and our earlier study using a rat model indicated that oxidative stress plays an important role in this process. Proteases are important mediators of cellular damage and are known to be activated in oxidative stress. This study looked at protease activity in enterocytes after surgical stress. METHODS: Surgical stress was induced by opening the abdominal wall and handling the intestine as done during laparotomy, in normal and xanthine oxidase-deficient rats. Enterocytes at various stages of differentiation were isolated and protease activity and protection offered by xanthine oxidase inhibitors were determined. Mitochondria and cytosol were prepared from total isolated enterocytes at different periods after surgical stress and protease activation was studied. RESULTS: Surgical stress induced activation of proteases in both the villus and crypt cells. Protease activation is seen in both mitochondria and cytosol, and similar to the other alterations in mucosal cells, protease activation was maximum 60 min after stress, returning to normal by 24 h. Thiol compounds modulate protease activity in both mitochondria and cytosol and the activation is not seen in xanthine oxidase-deficient animals. CONCLUSIONS: Surgical stress induces activation of proteases in villus and crypt cells of the small intestine. Both mitochondrial and cytosolic proteases are activated and free radicals generated by xanthine oxidase may mediate protease activation after surgical stress in the intestine.     

自身免疫性睾丸炎对精子特异性酶和生育功能的影响

目的 :研究自身免疫性睾丸炎对精子特异性酶和生育功能的影响。　方法 :复制豚鼠实验性变态反应性睾丸炎 (EAO)模型 ,采用酶动力学分光光度法和明胶固定底物薄膜法 ,观察EAO状态下精子顶体蛋白酶、透明质酸酶、精子胞质乳酸脱氢酶、附睾尾部精子和睾丸组织形态的变化。　结果 :EAO造成附睾精子顶体酶系中顶体蛋白酶、透明质酸酶和乳酸脱氢酶活性下降、附睾尾部精子质量下降、睾丸生精细胞发生退行性病变。　结论 :EAO明显影响雄性豚鼠生育力 ,睾丸生精细胞和附睾精子可能是主要作用环节。     

Protease activity as a prognostic factor for wound healing in complex wounds

Healing mechanisms are disrupted in complex wounds. Proteases may persist longer in nonhealing wounds. We sought to investigate whether protease activity, protease inhibitor activity, or their combinations are independent prognostic factors for healing of complex wounds. We searched MEDLINE, EMBASE, CINAHL, and The Cochrane Library to March 2019. Study selection comprised longitudinal studies assessing the independent effect of proteases, their inhibitors or ratios of the two, on healing of complex wounds, while controlling for confounding factors. Two reviewers independently extracted data and assessed risk of bias. We conducted meta‐analyses separately for proteases, inhibitors, and ratios. We graded the evidence certainty (quality). We identified eight eligible studies in 10 cohorts involving 343 participants. Risk of bias was moderate or high. Elevated protease activity may be associated with less wound healing (standardized mean difference [SMD]: ?0.41, 95% CI ?0.72 to ?0.11; nine cohorts); and elevated protease inhibitor activity with more healing (SMD: 0.37, 95% CI 0.06‐0.68; five cohorts), this is low certainty evidence. Increased protease: inhibitor ratios may be associated with less healing (SMD ?0.47, 95% CI ?0.94 to ?0.01; four cohorts), but this evidence is of very low certainty. Heterogeneity in protease activity was unexplained by prespecified subgroup analyses for wound type or protease activity status, but partially explained by protease class. Posthoc analysis suggested elevated levels of a particular protease, MMP‐1, may be associated with more healing and other proteases with less healing. This is low/very low certainty evidence. Limitations were small included studies at moderate or high risk of bias, and the use of posthoc analyses. Elevated protease activity and protease: inhibitor ratios may be associated with less healing, and elevated inhibitor levels with more healing. There may be important differences between MMP‐1 and other proteases. High quality research is needed to explore these new findings further.     

On the mechanisms of inhibition of insulin action by small-molecular-weight trypsin inhibitors

Evidence from a number of laboratories has suggested that the mechanism of insulin action involves the release of an intracellular mediator polypeptide from the plasma membrane. It has been proposed that activation of a protease with trypsin-like specificity is involved in release of the putative mediator. In an effort to assess the potential role of such a protease in intact cells, the present study tested the effects of a variety of low-mol-wt protease inhibitors on insulin's metabolic action in isolated rat epididymal fat cells. The protease inhibitors studied included p-aminobenzamidine, benzamidine, phenylguanidine, diisopropylfluorophosphate, leupeptin, and the competitive substrate N-alpha-tosyl-L-arginine methylester. Leupeptin was devoid of activity. Most of the other inhibitors used were able to interfere with insulin-stimulated metabolism if used in sufficiently high concentrations, concentrations considerably higher than those required for inhibition of known proteases or inhibition of intracellular processes in a previously described system which involves a trypsin-like enzyme. Moreover, they displayed various activities unrelated to protease inhibition that could explain their effects on insulin action better than protease inhibition. While none of the data on individual inhibitors were by themselves convincing enough to either confirm or reject the hypothesis concerning the involvement of a protease with trypsin-like specificity in insulin action, taken together our results do weaken the hypothesis considerably and in particular render the involvement of an extracellular trypsin-like enzyme improbable.     

Regulation of ENaCs by proteases: An increasingly complex story

Proteolytic processing of epithelial Na+ channel (ENaC) subunits has an important role in the regulation of ENaC gating. This Commentary addresses the potential roles of specific proteases and protease inhibitors in the control of Na+ channel gating.     

A locus on chromosome 20 encompassing genes that are highly expressed in the epididymis

During liquefaction of the ejaculate, the semen coagulum proteins semenogelin I （SEMG1） and semenogelin Ⅱ （SEMG2） are degraded to low molecular mass fragments by kallikrein-related peptidase 3 （KLK3）, also known as prostate-specific antigen. Semenogelin molecules initiate their own destruction by chelating Zn^2＋ that normally would completely inhibit the proteolytic activity of KLK3. In a similar way, semenogelins might regulate the activity of kallikrein-related peptidases in the epididymis, something that might be of importance for the maturation of spermatozoa or generation of anti-bacterial peptides. Studies on the evolution of semen coagulum proteins have revealed that most of them carry an exon that displays a rapid and unusual evolution. As a consequence, homologous proteins in rodents and primates show almost no conservation in primary structure. Further studies on their evolution suggest that the progenitor of the semen coagulum proteins probably was a protease inhibitor that might have displayed antimicrobial activity. The semenogelin locus on chromosome 20 contains at least 17 homologous genes encoding probable protease inhibitors with homology to semen coagulum proteins. All of these are highly expressed in the epididymis where they, similar to the semenogelins, could affect the maturation of spermatozoa or display antibacterial properties. （Asian J Androl 2007 July; 9： 540-544）     

Matrix metalloproteinases contribute to insulin insufficiency in Zucker diabetic fatty rats

To assess the molecular changes associated with pancreatic beta-cell dysfunction occurring during the onset of type 2 diabetes, we profiled pancreatic islet mRNAs from diabetic male and high-fat-fed female Zucker diabetic fatty (ZDF) rats and their nondiabetic lean counterparts on custom islet-specific oligonucleotide arrays. The most prominent changes in both the male and female models of type 2 diabetes were increases in the mRNAs encoding proteases and extracellular matrix components that are associated with tissue remodeling and fibrosis. The mRNAs for metalloproteinase (MMP)-2, -12, and -14 were sharply increased with the onset of islet dysfunction and diabetes. Zymography of islet extracts revealed a concurrent, >10-fold increase in MMP-2 protease activity in islets from 9-week-old male ZDF rats. Treatment of female ZDF rats receiving a diabetogenic diet with PD166793, a broad-spectrum MMP inhibitor, substantially prevented diabetes. The effect of this compound was due in part to marked beta-cell expansion. These studies indicate that MMPs contribute to islet fibrosis and insulin insufficiency in ZDF rats. Class-targeted protease inhibitors should be explored for their potential therapeutic utility in preservation of beta-cell mass in type 2 diabetes.     

Tissue Dissociation Enzyme Neutral Protease Assessment

Neutral proteases, essential components of purified tissue dissociation enzymes required for successful human islet isolation, show variable activities and effects of substrate on their activities. Initially we used a spectrophotometric endpoint assay with azocasein substrate to measure neutral protease activity. After critical review of the results, we observed these data to be inconsistent and not correlating expected differences in specific activities between thermolysin and Bacillus polymyxa proteases. This observation led to the development of a fluorescent microplate assay using fluorescein isothyocyanate-conjugated bovine serum albumin (FITC-BSA) as the substrate. This simpler, more flexible method offered a homogeneous, kinetic enzyme assay allowing determination of steady state reaction rates of sample replicates at various dilutions. The assay had a linear range of 4- to 8-fold and interassay coefficients of variation for B polymyxa protease and thermolysin of <9% and <15%, respectively, which were lower than those using the spectrophotometric endpoint assay, namely, 54% and 36%, respectively. This format allowed for incorporation of enzyme inhibitors, as illustrated by addition of sulfhydryl protease inhibitors, which, consistent with earlier reports, strongly indicated that the main contaminant in purified collagenase preparations was clostripain. Determination of the specific activities for several purified neutral proteases showed that the B polymyxa and Clostridium histolyticum proteases had approximately 40% and 15% specific activities, respectively, of those obtained with purified thermolysin, indicating the different characteristics of neutral protease enzymes for cell isolation procedures.     

Cold-induced apoptosis of rat liver endothelial cells: involvement of the proteasome

BACKGROUND: We have previously shown that in cultured liver endothelial cells the mere sequence of hypothermia and rewarming induces a pronounced, iron-dependent apoptosis that is likely to contribute to hepatic preservation injury. Here we study the involvement of proteases in this cold-induced apoptosis. METHODS: Cultured liver endothelial cells were incubated in preservation solutions at 4 degrees C in the absence or presence of protease inhibitors. Cell injury and different protease activities were assessed. RESULTS: Cold incubation of liver endothelial cells in University of Wisconsin or histidine-tryptophan-ketoglutarate (HTK) solution led to marked cell injury, which was strongly inhibited by the protease inhibitor 3,4-dichloroisocoumarin (DCI) (lactate dehydrogenase release: 86.0+/-2.6% in HTK and 4.0+/-0.4% in HTK + DCI after 24 hr of cold incubation). Determination of protease activity showed a doubling in the activity of a Suc-Leu-Leu-Val-Tyr-AMC-cleaving protease and some increase in a relatively low Suc-Ala-Ala-Ala-AMC-cleaving activity after 8 hr of cold incubation in HTK solution or 16 hr in University of Wisconsin solution. Further characterization of the protease activities showed that they belonged to two different proteases, with the major activity being calcium independent, inhibited by DCI, MG-132, and lactacystin, and strongly decreased by immunoprecipitation with an antiproteasome antibody. Preincubation of the cells with the iron chelator deferoxamine prevented the cold-induced increase of both activities. CONCLUSION: These results show that (i) the proteasome and possibly, in addition, a serine protease are involved in cold-induced apoptosis of liver endothelial cells and (ii) the protease activation is downstream from the increase in intracellular chelatable iron.