血小板活化因子对生育与不育男性精子顶体反应的影响

<正>在动物与人类受精过程中,获能和顶体反应(AR)是精子穿透卵子透明带,并与卵膜融合,最后发生受精的必要条件。只有获能的精子才能发生顶体反应。同样,只有经过顶体反应的精子才能与卵子受精[1]。已知大量的内源性因子可以调节精子     

孕酮对人精子受精能力的影响

用不同浓度孕酮处理人精子，研究孕酮对人精子穿透去透明带金黄地鼠卵能力的影响。结果发现低浓度孕酮使人精子的穿卵率和穿卵指数均有极显著的提高，而高浓度孕酮则对人精子穿卵率和穿卵指数有降低作用。     

孕酮对人精子受精能力的影响

用不同浓度孕酮（Ｐ）处理人精子，研究孕酮对人精子穿透去透明带金黄地鼠卵能力的影响。结果发现低浓度孕酮（２０μｍｏｌ／Ｌ）使人精子的穿卵率和穿卵指数均有极显著的提高，而高浓度孕酮（１０００μｍｏｌ／Ｌ）则对人精子穿卵率和穿卵指数有降低作用。提示人工授精时可用低浓度孕酮处理穿卵率低下的病人精子，以提高人工授精的成功率     

体外诱导人精子顶体反应方法的比较

目的 比较人透明带糖蛋白、孕酮和钙离子载体A23187在体外诱导人精子顶体反应的效率和可行性.方法 通过精子上游法制备正常男性精子悬液,分别以人透明带糖蛋白、孕酮和钙离子载体A23187诱导精子发生顶体反应.随后,运用考马斯亮蓝染色判断精子顶体的状态,并统计精子的顶体反应率.结果 人透明带糖蛋白、孕酮和钙离子载体A23187三法诱导的人精子顶体反应率分别为:(32.45±6.92)%(n=11)、(42.41±19.19)%(n=22)和(68.24±8.85)%(n=43),经统计学分析各组间差异均有统计学意义(P<0.05).结论 三种方法 中,钙离子载体A23187诱导效果最好,且不同个体间差异较小;而孕酮诱导的精子顶体反应率在不同个体间差异较大.     

不育男性精浆碳酸氢钠含量与精子活力等参数的关系

本文对80例不育症患者测定精浆N_aHCO_3含量,同时与粘液质量相比较。认为,精浆中N_aHCO_3含量与精子活力有着密切的关系,而与精液的PH值、粘稠度、精子密度无关,并就精浆N_aHCO_3含量测定在男子不育症诊治方面的意义进行探讨。     

不育男性精子形态与体外受精妊娠结局的相关性研究

目的 探讨不育男性精子形态与体外受精妊娠结局的关系.方法 按照WHO《人类精液及精子-宫颈粘液相互作用实验室检验手册》要求对573例接受辅助生殖技术治疗的男性不育患者的精子形态进行分析,根据患者正常精子形态百分比分为2组(＜1 5％:≥1 5％),分别采用体外受精(IVF)或卵泡浆内单精子注射(ICSI)的受精方式,观察精子形态对IVF及ICSI的受精率、卵裂率、优质胚胎率、妊娠率和流产率等的影响.结果采用IVF(327例)受精方式下,两形态组精子的受精率、卵裂率、优质胚胎率和妊娠率差异均有统计学意义(P＜0.05),但流产率差异无统计学意义(P＞0.05);而采用ICSI(246例)受精方式下,两形态组精子的上述指标比较差异均无统计学意义(P＞ 0.05).结论不育男性精子形态异常影响IVF治疗结局,但对ICSI治疗结局无影响,对于精子形态异常的患者应采用ICSI的受精方式.     

硝苯地平对人精子膜功能完整性及压力敏感性Ca~(2+)内流影响的实验研究

目的:探讨模拟正常血药浓度下硝苯地平体外对人精子膜功能完整性及压力敏感性Ca2+内流的影响。方法:将10例生育男性的精液经上游优化处理后,分别与20、100、20×103ng/ml的硝苯地平在体外共同孵育60min,同时设立对照组进行低渗肿胀(HOS)试验及测定压力导致的精子细胞内钙离子浓度的变化。结果:20×103ng/ml组的精子HOS百分率明显低于对照组,差异具有显著性(P<0.01)。20、100、20×103ng/ml组的精子细胞内Ca2+荧光差值均明显低于对照组,差异具有显著性(P<0.01)。结论:硝苯地平在体外可以改变精子膜功能完整性,抑制压力敏感性Ca2+内流。治疗剂量的硝苯地平在体外可影响精子质量。     

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     

蛙皮素对PC-3细胞骨架及细胞内游离Ca~(2+)的影响

目的:观察蛙皮素(BBS)对前列腺癌PC-3细胞骨架形态及细胞内游离Ca2+([Ca2+]i)浓度的影响。方法:①利用免疫荧光法(IH),结合激光扫描共聚焦显微镜(LSCM)检测10-5mol/L浓度BBS处理的PC-3细胞角蛋白(CK)的表达,以反映其对细胞骨架形态的影响;②应用Fluo-3/AM荧光标记技术和LSCM检测不同浓度BBS(10-9、10-7、10-5mol/L)处理的PC-3细胞[Ca2+]i浓度。结果:①10-5mol/L浓度的BBS可促进PC-3细胞CK表达及伪足形成;②BBS可提高PC-3细胞[Ca2+]i浓度,并具有浓度依赖性。结论:实验证明一定浓度的BBS可明显提高PC-3细胞[Ca2+]i浓度及CK表达,进而影响PC-3细胞骨架形态。本研究为探索BBS应用于肿瘤研究以及BBS作用后细胞内信息传递途径提供了基础。     

Effects of tumour necrosis factor alpha and interleukin-6 on progesterone and calcium ionophore-induced acrosome reaction

For human spermatozoa to successfully fertilize the oocyte, they need to undergo a timely acrosome reaction (AR). Factors which disturb the AR may lead to fertilization failure. The objective of this study was to investigate the effects of two cytokines namely tumour necrosis factor alpha (TNF-α) and interleukin-6 (IL-6) on the spontaneous, calcium ionophore-induced and progesterone-induced human sperm AR. Twenty-two normal semen samples were treated with increasing concentrations of TNF-α and IL-6 after spermatozoa were isolated by a double wash swim-up method. The AR was induced by calcium ionophore A23187 and progesterone. The AR was determined by using fluorescein isothiacyanate Pisum sativum agglutinin and observed under fluorescence microscope. Both TNF-α and IL-6 could decrease the spontaneous, ionophore and progesterone-induced AR ( p  <   0.05) in a dose-dependent manner. TNF-α showed a more potent inhibiting effect than IL-6 by inhibiting the AR at lower concentrations. This study has demonstrated that TNF-α and IL-6 play a role in inhibiting both the non-physiological as well as physiologically elicited AR by calcium ionophore and progesterone respectively.     

正常生育男性成熟精子mRNA的种类和特征

目的:剖析具有正常生育能力男性成熟精子mRNA的种类和丰度。方法:健康并育有后代的成年男性10例,禁欲1周后取精液,上游法优化精子,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)。结果:所建SAGE文库共获877个克隆,测序得到21 052个标签,出现两次以上的独特性标签有2 712种。经与SAGEm ap数据库比对,19.7%的独特性标签没有基因匹配,代表新基因;其余能匹配的基因中,67%具有蛋白质结合或核酸结合能力,41%具有催化活性,13%与信号转导有关,与细胞运输、精子结构、转录调节相关者分别达到10%左右。结论:人成熟精子中存在数量庞大、种类繁多的mRNA。     

Low-voltage-activated calcium channels are not involved in capacitation and biological response to progesterone in human sperm

The involvement of voltage-dependent calcium channels in the biological effects exerted by progesterone (P) on human spermatozoa is still a controversial issue. We have investigated the involvement of T-type calcium channels [voltage-operated calcium channels (VOCCT)] in two biological functions of human sperm, responsiveness to P and capacitation, by employing three different pharmacological antagonists of VOCCT, namely mibefradil (Ro 5967), pimozide and amiloride. Intracellular calcium [Ca(2+)]i increase in response to P was essentially unaffected by pre-treatment with mibefradil and pimozide at concentrations previously shown to prevent [Ca(2+)]i increase in response to zona proteins. Amiloride could not be tested in these experiments because it was found to interfere with fura-2 fluorescence. The increase in tyrosine phosphorylation stimulated by P in a protein of about 97 kDa was unaffected by the three antagonists. Acrosome reaction (AR) induced by P was also unaffected by mibefradil or pimozide but was significantly inhibited by amiloride at high concentrations (100 and 500 but not 10 microM). At 100 and 500 microM amiloride also inhibited Na/H exchanger as assessed by a fluorimetric method. We conclude that VOCCT are not involved in calcium increase and AR stimulated by P in human sperm. We next investigated the effect of the three VOCCT inhibitors on sperm capacitation by evaluating tyrosine phosphorylation and AR in basal conditions and in response to P. We found that the presence of pimozide and amiloride during capacitation stimulated a higher increase of tyrosine phosphorylation, whereas mibefradil was less effective. The ability of P to induce the AR, considered an index of occurrence of capacitation, was not affected by pimozide and mibefradil, whereas was inhibited by amiloride at concentrations that inhibit Na/H exchanger. In conclusion, our results do not support a major role of low-voltage-activated calcium channels in capacitation and response to P of human spermatozoa.     

Progesterone effect mediated by the voltage-dependent calcium channel and protein kinase C on noncapacitated cryopreserved bovine spermatozoa

An increase in intracellular calcium is essential to trigger capacitation and the acrosome reaction. The aim of this study was to determine the progesterone effect mediated by the voltage-dependent calcium channel and protein kinase C on heparin-capacitated and noncapacitated spermatozoa. Protein kinase C was activated by 1-oleoyl-2-acetyl glycerol, a membrane-permeant diacyl-glycerol, and inhibited by GF-109203X. The percentage of true acrosome reaction was evaluated using differential-interferential optical contrast microscopy and trypan blue stain. The calcium concentration was evaluated by FURA-2AM and methoxyverapamil was used as a voltage-dependent calcium channel inhibitor. A rapid calcium increase and acrosome reaction were induced by progesterone in capacitated and noncapacitated spermatozoa, a higher intracellular calcium increase being observed in capacitated than in noncapacitated samples (P < 0.05). The calcium increase and acrosome reaction were blocked significantly by GF-109203X in noncapacitated and capacitated spermatozoa by the addition of progesterone and/or 1-oleoyl-2-acetylglycerol. Methoxyverapamil blocked calcium influx in samples treated with progesterone and heparin/progesterone, but not in those treated with 1-oleoyl-2-acetyl glycerol. Progesterone induces the acrosome reaction in noncapacitated cryopreserved bovine spermatozoa through intracellular mechanisms dependent on protein kinase C and the voltage-dependent calcium channel.     

Reactive oxygen species influence the acrosome reaction but not acrosin activity in human spermatozoa

It is now widely accepted that the higher levels of reactive oxygen species (ROS) produced by damaged or deficient spermatozoa are associated with a loss of motility and a decreased capacity for sperm-oocyte fusion. Furthermore, earlier studies show, under physiological conditions, that some ROS may be involved in capacitation and hyperactivation of human spermatozoa. We measured ROS levels, acrosome reaction (AR) and acrosin activity (AA) in semen samples from suspected subfertile men to reveal the influence of ROS on AR and AA of human spermatozoa. Semen samples were obtained from 60 patients. Samples with > or = 1 x 10(6) leukocytes/mL were excluded from the study. ROS production was determined using a chemiluminescence technique. AR was determined using a triple stain technique. The percentage of acrosome-reacted spermatozoa after low temperature induction of the AR (test value), and the inducibility of AR (= the difference between the test value and the control), were calculated. The AA was analysed by determining the proteolytic potential of spermatozoa on gelatin plates. The mean halo diameter and percentage of halo formation in each sample were measured as AA parameters. Scatter plots of ROS levels and AR parameters showed that the percentage of acrosome reacted spermatozoa and AR inducibility were better in samples with low rather than high ROS levels. On the other hand, there were no apparent similarities between ROS and the AA parameters. Therefore, the percentage of acrosome-reacted spermatozoa and AR inducibility were significantly higher in the low than in the high ROS group (p = 0.028, p = 0.0001, respectively). In addition, there was no significant difference in AA parameters between groups. These findings suggest that lower ROS in semen may have a role in AR but excessive ROS may exert a negative influence on AR, while ROS in semen has no relationship to AA.     

胆囊结石病人胆囊胆汁中游离Ca^2＋升高

胆囊结石成因中钙非常重要，本研究旨在了解胆囊结石病人与非结石病人间睑汁中是否钙有量的不同。术中穿刺胆囊从57例病人抽取胆汁，其中22例病人无胆囊结石，19例为胆固醇结石，16例为胆色素结石，测定胆汁游离Ca2+，胆色素结石病人为1．79±0．44mmol／L，胆固醇结石病人为1．31±0．32mmol／L，非结石病人为1．05±0．10mmol／L。明石病人与非结石病人间测定值有明显的重叠，胆色素组与胆固醇组间P＜0．05，胆色素结石组与非结石组间P＞0．05，其他组间P＞0．05。这种异常变化与胆囊结石形成有关。     

电压依赖性钙离子通道基因在慢性非细菌性前列腺炎大鼠前列腺组织中的表达及其意义

目的 探讨电压依赖性钙离子通道(VDCC)在慢性非细菌性前列腺炎(CABP)发病机制中的作用.方法 40只雄性Wistar大鼠随机分成CABP模型组和对照组,采用去势加皮下注射雌二醇[O.25mg/2ml·k~(-1),qd×30d]的方法建立CABP大鼠模犁.造模成功后取两组大鼠的前列腺组织,最后应用荧光定量反转录酶-聚合酶链式反应(RT-PCR)技术分别检测20例CABP组和20例对照组大鼠前列腺组织中各类型VDCC的a_1mRNA的表达量.结果 与正常组Wistar大鼠相比,CABP组Wistar大鼠前列腺组织中VDCC的a_1G(0.30+0.27)、a_1H(1.45:1:2.30)mRNA表达的差异具有统计学意义·(P<0.05).结论 T型VDCC的基因表达异常可能是导致CABP原因之一.     

生育与不育男性精浆总抗氧化能力分析

目的:分析生育与不育男性精浆中总抗氧化能力(TAC)及其在男性生育中意义。方法:225例男性不育患者分为6组,分别为:梗阻性无精子症组(n=10),非梗阻性无精子症组(n=42),少精子症组(n=20),弱精子症组(n=78),少弱精子症组(n=57),以及正常精子症组(n=18)。28例正常生育男性作为对照(生育组)。分别采用计算机辅助精液分析(CASA)系统进行精液参数分析,采用比色法检测精浆TAC水平。结果:生育组男性精浆TAC为(19.82±6.33)U,梗阻性无精子症组(1.71±1.33)U,非梗阻性无精子症组(12.73±9.44)U,少精子症组(10.85±6.64)U,弱精子症组(13.88±8.24)U,少弱精子症组(11.20±7.02)U,正常精子症组(18.07±8.73)U;与生育组精浆TAC[(19.82±6.33)U]相比,在各不育症组中,除正常精子症组精浆TAC与生育组差异无显著性外,其余各组均显著低于生育组(P<0.01)。精浆TAC与精子密度(r=0.182,P<0.05)和a级精子(r=0.150,P<0.05)呈显著正相关。结论:精浆中TAC水平与男性不育密切相关,精浆中过低的TAC水平可能是引起男性不育的病因之一。