干扰素-γ和肿瘤坏死因子-α对精子受精能力的影响及机制初探

目的:观察IFN-γ和TNF-α对正常精子顶体酶活性和顶体反应率的影响,并对其变化机制进行初步探讨。方法:36例精液常规分析基本正常标本,经75%Percoll分离后,分别或联合使用IFN-γ和TNF-α处理(终浓度均为30ng/ml)。采用BAEE/ADH联合法测定精子顶体酶活力的变化。用三色染色法观察精子顶体反应率的变化。用高效液相色谱法(HPLC)检测精子NO含量。用试剂盒法测定Na+-K+-ATPase、Ca2+-ATPase和SOD活性。结果:IFN-γ和TNF-α单独及联合使用时,均可显著降低精子顶体酶活性和顶体反应率(P<0.05或P<0.01),且TNF-α的抑制作用更强些;IFN-γ可使精子Na+-K+-ATPase、Ca2+-ATPase和SOD活性显著降低(P<0.01),且两者协同作用更低,但TNF-α对Na+-K+-ATPase和Ca2+-ATPase活性几乎无作用;IFN-γ、TNF-α及两者合用时均使精子NO含量显著增加(P<0.01)。结论:IFN-γ和TNF-α对精子顶体酶活性及顶体反应率有一定的抑制作用,并且可能通过对精子Na+-K+-ATPase、Ca2+-ATPase、SOD活性以及NO含量等多方面影响来实现的。     

肿瘤坏死因子对人精子顶体酶和顶体反应的影响及机制探讨

目的 研究肿瘤坏死因子(TNF-α)对正常精子顶体酶活性和顶体反应的影响及其机制。方法采用BAEE/ADH联合法测定顶体酶和三色染色法技术测定顶体反应。结果 TNF-α可显著降低精子顶体酶的活性和顶体反应(P<0.01;P<0.01),并且它可使精子中的Ca2+-ATPase和SOD的活性显著降低(P<0.05;P<0.001);但TNF-α可使精子中NOS活性增强及NO含量增加(P<0.001;P<0.001);对Na+-K+-ATPase活性影响不明显(P>0.05)。结论 TNF-α对精子顶体酶及顶体反应有一定的抑制作用,并且可能通过降低Ca+-ATPase活性,自由基和NO及增加NOS等多种途径来实现的。     

NO通过人精子顶体酶对顶体反应的影响

目的探讨NO通过精子顶体酶(acrosin)对项体反应(acrosome reaction,AR)的影响。方法采用BAEE/ADH法测定精子顶体酶的活性,以及通过FITC-PSA法检测精子顶体反应。结果NO供体SNP可诱导人精子表达顶体酶活性,同时促进入精子顶体反应；顶体酶抑制剂TLCK能抑制顶体酶活性,并可抑制SNP诱导的人精子顶体反应。结论NO可能通过调节人精子顶体酶的活性而诱导精子的顶体反应。     

蛋白激酶C对精子活力和顶体反应的影响

蛋白激酶C(PKC)位于精子头部赤道段和尾部主段。外源性PKC激动剂可增强精子鞭毛的活力 ,而PKC抑制剂如癌基因抑活药 (staurosporine)则能抑制这种作用。精子中PKC的含量与精子活力呈显著正相关 (r =0 .9,P <0 .0 0 1)。精子与透明带 (ZP)结合刺激顶体反应的发生 ,导致顶体释放多种水解酶以及精子暴露出新的膜区域。ZP与精子质膜上的受体结合后 ,可以调节腺苷酸环化酶 (AC)的活性 ,使cAMP浓度升高并激活蛋白激酶A(PKA)。PKA能激活位于顶体外膜的电压依赖性钙通道 ,后者使顶体内部的Ca2 + 释放至胞质中 ,磷脂酶C(PLC)因Ca2 + 浓度升高而激活并水解磷脂酰肌醇二磷酸 ,其水解产物激活PKC ,后者使精子质膜上电压依赖性钙通道 (L型 )开放 ,胞质中第二次较大幅度Ca2 + 浓度的上升导致质膜溶解及顶体反应发生。推测PKC参与调节精子的活力和顶体反应     

精子诱发顶体反应与精子顶体酶活性测定关系的研究

目的 通过对精液常规正常的不育男子检测人类精子诱发顶体反应和测定精子顶体酶活性,探讨两种检测结果 的相关性.方法 40例精子密度≥20×106/ml、活动力(a+b)级精子≥50%的不育男子精液,采用离子载体A23187诱导精子顶体反应(AR),用FITC-PSA荧光染色法分析AR,并计算顶体反应发生率;用分光光度比色法(Kennedy法)测定精子顶体酶活性.用组间方差分析方法 比较精子顶体反应发生率与精子顶体酶活性之间关系.结果 两种检测结果 没有明显相关性(r=0.292,P>0.05).结论 两种检测联合应用可作为临床分析男性不育病因的检测手段.     

γ-氨基丁酸对精子顶体酶活性的影响

目的 :研究γ 氨基丁酸 (GABA)对生育男性精子及抗精子抗体 (AsAb)阳性病人的精子顶体酶活性的影响。　方法 :用BAEE ADH联合法同时测定GABA对精子顶体酶活性的影响。　结果 :GABA可明显提高正常及AsAb阳性精子顶体酶的活性 (P <0 .0 1) ;并使精子的Na+ K+ ATPase、Ca2 + ATPase、超氧化物歧化酶 (SOD)的活性增加(P <0 .0 1,P <0 .0 5 ,P <0 .0 0 1)。　结论 :GABA对精子顶体酶活性有显著影响     

低氧对大鼠精子体外顶体反应和顶体酶、透明质酸酶活性的影响

目的研究低氧对大鼠精子顶体酶、透明质酸酶活性的影响以及对精子体外顶体反应发生的影响。方法成年Wistar大鼠随机分为4个组:常氧对照组、低氧5d组、低氧15d组和低氧30d组。低氧组置低压舱内模拟高原5000m,分别低氧5、15和30d。采用明胶薄膜法、透明质酸底物转化法、溶血磷脂胆碱(LPC)诱导金霉素荧光染色法,测定低氧对附睾尾精子顶体酶、透明质酸酶活性和体外顶体反应发生率的影响。结果低氧5、15和30d组大鼠精子顶体反应发生率,与常氧对照组相比均显著降低,分别由常氧对照组的(30.5±3.5)%,下降至(11.7±0.9)%(P<0.01)、(10.8±1.0)%(P<0.01)和(10.0±1.4)%(P<0.01);低氧5、15和30d组大鼠精子顶体酶阳性率降低,分别由常氧对照组的(81.67±7.16)%,下降至(54.17±3.82)%(P<0.01)、(30.00±3.92)%(P<0.01)和(43.00±3.63)%(P<0.01);低氧各组透明质酸酶活性无显著改变(P>0.05)。结论低氧抑制了精子顶体酶活性和顶体反应发生。     

人精浆抗精子抗体与顶体酶活性关系的探讨

目的 :研究精浆抗精子抗体 (AsAb)对精子顶体酶活力的影响。　方法 :用间接血凝法测定 34 32例不育男性和 6 5例生育男性精浆的AsAb ,并对其中的 2 882例不育男性精子用比色法进行了顶体酶活性的测定。　结果 :34 32例不育者精浆AsAb阳性率为 10 .2 0 %,2 882例进行了顶体酶活性测定的不育者精浆AsAb阳性率为 9.37%,对照组精子顶体酶活性明显高于各不育组 (P <0 .0 0 1)。不育组AsAb阳性组与AsAb阴性组比较顶体酶活性没有明显差异 (P >0 .0 5 ) ,顶体酶活性正常与异常组AsAb阳性率比较差异无显著性 (P >0 .0 5 )。　结论 :精浆AsAb对精子顶体酶活性没有影响。     

血小板活化因子对精子功能的影响

血小板活化因子(PAF)是一种内源性脂质分子,除具有血小板激活功能外,还具有多效生物学特性。近年来发现,PAF对男性生殖和精子功能起重要作用:通过细胞上特异的G蛋白受体介导的三磷酸肌醇二酰甘油途径使胞内Ca2+浓度升高而间接影响精子功能。精源性PAF浓度能反映精子活力和受精潜能,可作为临床评估男性生育力的指标;外源性PAF能提高精子活力、顶体反应及人工授精的妊娠率。     

几种离子与人类精子顶体反应的关系

本文用离子载体A23187诱导人类精子的顶体反应,并通过去除介质中Na~+,或在介质中加入K~+阻滞剂,或Ca~(2+)阻滞剂,或Na~+-K~+-ATP酶阻滞剂的方式来研究人类精了顶体反应与几种离子及Na~+泵的关系。结果表明:在人类精子顶体反应时,要有Na~+,K~+,Ca~(++)及Na~+泵的参与。但是,Ca~(++)进入精子的途径可能不是Ca~(++)的通道,     

男性不育患者精液质量与精子顶体酶活性关系分析

目的:分析男性不育患者精液参数与精子顶体酶活性变化,探讨精液质量与顶体酶活性的关系。方法:对214例男性不育患者的精液作精子顶体酶活性、弹性蛋白酶、果糖、α葡糖苷酶、锌、酸性磷酸酶、精子尾部低渗肿胀试验检测,同时精子质量检测仪作精液分析。以检出精子顶体酶活性正常的(48.2~218.7μIU/106精子)111例作为对照组,与顶体酶活性异常(<48.2μIU/106精子)的103例精液参数作对比分析。结果:两组精液参数的精子密度、精子活动率、a+b级精子百分率、精子尾部低渗肿胀试验差异均有非常显著性(P<0.001)。弹性蛋白酶差异有显著性(P<0.05)。果糖、α葡糖苷酶差异亦有显著性(P<0.05)。精液量、锌、酸性磷酸酶差异无显著性(P>0.05)。结论:精子顶体酶活性与精液质量关系密切,精子顶体酶活性是反映精液质量的一项可靠指标。     

Assessment of released acrosin activity as a measurement of the sperm acrosome reaction

Aim： To develop a method for assessing sperm function by measuring released acrosin activity during the acrosome reaction （AR）. Methods： Human semen samples were obtained from 24 healthy donors with proven fertility after 3-7 days of sexual abstinence. After collection, samples were liquefied for 30 min at room temperature. Standard semen parameters were evaluated according to World Health Organization （WHO） criteria. Calcium ionophore A23187 and progesterone （P4） were used to stimulate the sperm to undergo AR. After treatment, sperm were incubated with the supravital dye Hoechst33258, fixed in a glutaraldehyde-phosphate-buffered saline solution, and the acrosomal status was determined by fluorescence microscopy with fluorescein isothiocyanate-labeled Pisum sativum agglutinin （FITC-PSA）. The percentage of sperm undergoing AR （AR%） was compared to sperm acrosin activities as assessed by spectrocolorimetry. The correlation between AR% and acrosin activity was determined by statistical analysis. Results： The AR% and released acrosin activity were both markedly increased with A23187 and P4 stimulation. Sperm motility and viability were significantly higher after stimulation with P4 versus stimulation with A23187 （P 〈 0.001）. There was a significant positive correlation between released acrosin activity and AR% determined by FITC-PSA staining （r= 0.916, P 〈 0.001）. Conclusion： Spectrocolorimetric measurement of released acrosin activity might serve as a reasonable alternative method to evaluate AR. （Asian J Androl 2008 Mar, 10： 236-242）     

Percoll优选对形态正常精子百分率和顶体酶活性的影响

目的 :了解形态正常精子百分率和顶体酶活性在Percoll优选前后的变化。　方法 :30例不育男性精液标本 ,应用计算机精液分析仪进行精子形态分析 ,分光光度比色法检测顶体酶活性。　结果 :Percoll优选后形态正常精子百分率和精子顶体酶活性均明显高于Percoll优选前 (P均 <0 .0 0 1)。　结论 :Percoll法优选精子后形态正常精子百分率和顶体酶活性增加。     

A simple, clinical assay to evaluate the acrosin activity of human spermatozoa

Acrosin, a sperm-specific acrosomal proteinase, has an essential role in the fertilization process. Low levels of acrosin appear to be associated with subfertility and infertility, and the acrosin activity of spermatozoa may potentially be a useful indicator of semen quality. The standard acrosin tests employed by research laboratories are too complicated and/or time consuming for clinical use; therefore, a simple assay has been developed to assess total acrosin activity (acrosin and activatable proacrosin). To perform the test, liquefied semen is centrifuged over Ficoll, the washed sperm pellet is suspended in a detergent (Triton X-100)-substrate (N-alpha-benzoyl-DL-arginine p-nitroanilide) buffer, pH. 8.0, and the amidase activity is determined spectrophotometrically after a 3-hour incubation period. Amidase activity can be inhibited with benzamidine, indicating that the activity is primarily or entirely due to acrosin. The absence of detergent in the incubation medium results in greatly reduced activity. The assay is repeatable, linear with increasing sperm concentration, sensitive to a lower limit of 2 x 10(6) spermatozoa, and the results correspond to those obtained with a standard acrosin extraction and assay technique. Storage of ejaculates at 3 to 6 C or at 22 to 24 C for 24 hours does not affect the acrosin activity significantly but much higher temperatures can cause a loss of activity. Freezing ejaculates results in a large decrease in sperm acrosin activity. Leukocytes show minimal activity in the assay. Sperm populations prepared by a swim-up procedure average approximately a 2-fold higher acrosin activity than the original ejaculates. Preliminary experiments indicate that the average sperm acrosin activity of ejaculates whose spermatozoa successfully fertilize human eggs in vitro is significantly higher than those that do not fertilize eggs.     

Determination of sperm acrosin activity for evaluation of male fertility

Aim: To investigate a simple method for assaying acrosin activity for the evaluation of male fertility. Methods:The acrosin activity of 7.5 × 10~6 sperm without seminal plasma and acrosin activity inhibitors was assayed using N-α-benzoyl-DL-arginine-p-nitroanilide (BAPNA) and detergent (Triton X-100) as substrate. Results: The acrosin ac-tivity of 60 normal fertile men (35 ± 10 μIU/10~6 sperm ) was higher than that of 168 infertile men ( 16 ± 8 μIU/10~6sperm) (P <0.01). It was indicated that there was a significant positive correlation between the acrosin activity andthe sperm motility ( r ≥ 0.6534, P < 0.01) and a significant negative correlation between the sperm malformed rateand the WBC number ( r ≤ -0.5426, P < 0.01). The temperature and time of incubation and the sperm concentrationcould influence the assay results. Conclusion: Acrosin activity is an important index for the evaluation of male fer-tility. The approach developed by the authors is a simple method for the determination of acro     

KF-950对人精子顶体素活性及顶体的影响

目的 :研究顶体素抑制剂 4 胍基苯甲酸盐 (KF 95 0 )对人精子顶体素的抑制作用和对顶体的影响。　方法 :醋酸抽提并纯化人精子顶体素 ,BAEE/ADH法测定不同剂量KF 95 0作用后残余酶的活性。以生物素标记的豌豆凝集素为探针 ,采用ABC染色法观察KF 95 0对完整精子顶体的影响。　结果 :①正常人精子顶体素活性为(37.6 5± 4 .4 7)U/L(pH 8.5 ,2 5℃ ) ;②残余酶活性与KF 95 0剂量呈负相关 (r =- 0 .998) ,并呈量 效依赖关系 ,回归方程 :Y =7.5 7- 1.895X ;③随药物浓度的增加和作用时间的延长 ,顶体完整着色率均明显下降 (P <0 .0 1)。结论 :KF 95 0能直接抑制顶体素活性 ,推测对精子顶体也有明显的破坏作用 ,是一种新型、高效的精子顶体素抑制剂。     

不育患者精浆α-1,4糖苷酶活性与精液参数的关系

目的　探讨精浆 α-1 ,4糖苷酶活性与精液参数之间的关系。　方法　分光光度比色法测定精浆 α-1 ,4糖苷酶活性及进行精液常规分析。　结果　 2 90 2例男性不育者精浆α-1 ,4糖苷酶活性异常率为 3 8.87%。该酶活性与精子密度、精子活率、a,b级精子活力和顶体酶活性呈显著正相关 ( r分别为 0 .460、0 .1 2 2、0 .0 86和 0 .2 3 0 ,P均 <0 .0 0 1 ) ,而与精液量、精液 p H、液化时间和畸形精子率无显著相关 ( P>0 .0 5)。 α-1 ,4糖苷酶活性正常组精子密度、活率、a,b级精子活力和精子顶体酶活性均明显高于 α-1 ,4糖苷酶活性异常组 ( P<0 .0 0 1 )。以常规精液分析法 ( RSA)主要参数正常与否分成的两组间α-1 ,4糖苷酶活性差异有显著性 ( P<0 .0 0 1 )。　结论　α-1 ,4糖苷酶活性对精子密度、活率、a,b级精子活力和顶体酶活性均有明显影响 ,对精液量、精液 p H、液化时间和畸形精子率无显著影响