氰戊菊酯体外对大鼠精子运动能力的影响

目的:观察氰戊菊酯(Fen)对大鼠精子运动能力的直接作用。方法:收集7只健康成年雄性SD大鼠附睾尾精子,用Fen进行体外染毒,剂量分别为1、4、16、64μmol/L,以Fen溶剂(二甲亚砜)作为对照组。在染毒1、2、4 h后用计算机辅助精子分析(CASA)系统对精子运动速度[曲线运动速度(VCL)、直线运动速度(VSL)、平均路径速度(VAP)、鞭打频率(BCF)]和运动方式[前向性(STR)、直线性(LIN)]进行检测,观察Fen对离体大鼠精子运动能力的影响。结果:Fen染毒1、2 h后,可见64μmol/L组的VSL、BCF、LIN和STR与对照组相比差异有显著性(P<0.01或P<0.05)。染毒4 h后,除上述指标外,VCL在16、64μmol/L组与对照组相比也呈现出明显的下降(P<0.01)。时间-效应关系分析显示,Fen(16、64μmol/L)染毒4 h组与染毒1 h组相比,VCL和STR显著下降(P<0.01或P<0.05)。结论:Fen可作用于大鼠附睾尾精子,并对大鼠精子运动有直接毒性效应。     

线粒体氧化磷酸化抑制剂FCCP体外对人精子动力学和线粒体功能的影响

目的通过线粒体氧化磷酸化(OXPHOS)特异性抑制剂FCCP对人精子活动力及其线粒体功能影响的研究,探讨氧化磷酸化在精子能量代谢中的作用。方法选择来自捐精志愿者的正常精液8份,优选后制备精子悬液,将每份精子悬液分为4组,分别与终浓度为0μmol/L(对照组)、2.5μmol/L、5μmol/L和10μmol/L的FCCP共孵育1h、3h、5h,以精子动力学参数、线粒体膜电位、精子细胞内ATP含量、精子质膜完整性作为评价指标,分析比较各组间的差异。结果 (1)各组精子活动率和其他各项运动参数随着FCCP浓度增高呈下降趋势:孵育1h后,与对照组相比,仅10μmol/L组的精子活动率、前向运动百分率和精子头侧摆幅度(ALH)显著下降(P0.05),其余指标无显著性变化,而其余浓度处理组的各指标变化均无统计学意义(P0.05);孵育3h后,10μmol/L组的精子活动率、前向运动百分率、平均路径速率(VAP)、直线速率(VSL)、曲线速率(VCL)、ALH和鞭打频率(BCF)均显著降低(P0.05),5μmol/L组的ALH和BCF指标显著下降(P0.05);孵育5h后,与对照组相比,10μmol/L组的前述指标继续显著性下降(P0.05),5μmol/L组的精子活动率和前向运动百分率也出现显著降低(P0.05),而2.5μmol/L组各指标差异均无统计学意义(P0.05)。(2)精子线粒体膜电位(MMP)和ATP含量随FCCP浓度增加逐渐降低,10μmol/L组的MMP和ATP含量显著低于对照组(P0.05)。(3)质膜完整性比较中,10μmol/L组比对照组显著降低(73.94%vs.84.53%)(P0.05)。(4)随着FCCP孵育时间延长,各组精子活动率和前向运动百分率呈下降趋势。结论不同浓度的FCCP体外处理精子后,精子活动率和其他各项运动参数,以及反映线粒体活性的线粒体膜电位和ATP含量呈浓度依赖性下降,FCCP所抑制的线粒体氧化磷酸化是精子能量代谢的重要途径。     

人参皂甙Rb1促进大鼠雪旺细胞增殖的实验研究

目的：观察人参皂甙Rb1对体外培养的大鼠坐骨神经经雪旺经细胞增殖能力的影响，探讨其促进神经再生的作用机制。方法：取SD雄性大鼠坐骨骨神经体外培养第2代第5天，加入不同浓度的人参皂甙Rb1，利用MTT比色分析，^3H－胸腺嘧啶核甙测定法检测不同浓度人参皂甙Rb1在不同培养时间对体外培养大鼠旺细胞增殖的影响。结论：人参皂甙Rb1在10μg/ml的浓度对雪旺细胞增殖有明显促进作用，高浓度的人参皂甙Rb1 1mg/ml则显示抑制作用。而200μg/ml人参皂甙Rb1对细胞增殖的促进作用与对照组相近。结论：人参皂甙Rb1在适当浓度范围内可以促进雪旺细胞的增殖，从而为促进活体神经损伤的修复途径提供了一些研究基础。     

白藜芦醇对环磷酰胺诱导的人精子氧化损伤的保护作用乜照燕1,张亚楠2,花蕊2,陈丽君1,李亮1,吴海峰

目的 探讨白藜芦醇(RES)对环磷酰胺诱导的人精子氧化损伤的保护作用。方法 收集2018年1月至4月在河北医科大学第四医院生殖医学中心就诊男性患者的正常精液标本。将用密度梯度离心法处理后的正常精液标本用不同浓度(0、1.0、2.5、5.0、7.5μmol/L)环磷酰胺活性代谢产物4-过氧化氢环磷酰胺(4-HC)对精子进行体外毒性试验,继续培养24h,通过精子活力和精子存活率筛选4-HC作用半抑制浓度(IC50)。将处理后的正常精液用不同浓度RES(5、10、25、50、75μmol/L)预处理2h,再加入IC50浓度4-HC,测定精子活力和精子存活率选择RES最佳保护浓度。选择4-HC和RES浓度后,该实验分为四组:对照组(CON)、环磷酰胺组(4-HC)、环磷酰胺+白藜芦醇组(4-HC+RES)、白藜芦醇组(RES),检测各组精子活力、活率、氧化应激指标以及精子凋亡指标。结果 随4-HC浓度增加,精子活力和存活率降低,呈剂量依赖关系,依据IC50,2.5μmol/L4-HC为后续实验药物浓度。RES预处理后,精子活力和精子活率均得到改善,50μmol/L RES效果最佳,确定为后续实验药物浓度。结果显示:4-HC组较CON组,精子活力和存活率下降,氧化应激水平上升,精子凋亡增加,4-HC+RES组与4-HC组相比精子活力和存活率升高,氧化应激水平降低,精子凋亡降低。结论 RES可以减少化疗药物导致的精子氧化应激水平增加,能预防和缓解化疗药物对人精子造成的损伤。     

刺五加注射液与茶碱、咖啡因体外对精子运动功能影响的比较研究

目的:比较刺五加注射液与茶碱、咖啡因体外对精子运动功能的影响。方法:由12例弱精子症患者通过手淫获得并经上游优化处理的精子分别与一定浓度的刺五加注射液(10g/L)、咖啡因(7mmol/L)和茶碱(3mmol/L)一起孵化0h、1h、3h后,采用计算机辅助精液分析系统(CASA)检测精子的运动参数(精子活动率、前向性运动百分率、直线运动速度、曲线运动速度)。结果:刺五加注射液在体外能显著提高人精子活动率,前向性运动精子百分率,精子直线运动速度和曲线运动速度,其改善精子运动功能优于茶碱和咖啡因,差异均有显著性(P<0.05)。结论:中药刺五加注射液在体外能显著改善人精子的运动功能。     

生脉注射液在体外对精子运动能力和存活时间的影响

目的:观察生脉注射液对人精子运动参数和存活时间的影响。方法:将33例正常生育男性精液均分为两份,一份与生脉注射液+Hams-F10孵育,一份与Hams-F10共孵育。于0.5、1、2、4、8、12 h取样,检测精子存活率、并应用计算机辅助精子分析系统(CASA)测定精子的直线运动速度(VSL)、曲线运动速度(VCL)、平均路径速度(VAP)和头部侧摆幅度(ALH)等精子运动参数。结果:生脉组在孵育8 h时VCL明显高于Hams-F10组(P<0.05),12 h时生脉组VCL显著增高(P<0.01)。孵育4、8、12 h,生脉组VSL和VAP明显高于Hams-F10组(P<0.05)。孵育2、4、8、12 h时生脉组ALH明显高于Hams-F10组(P<0.05)。两份精液各时间段的精子死活比率相比,除12 h时有明显差异(P<0.05)外,其他各时间段无明显差异。结论:生脉注射液能提高精子的运动能力、延长精子的体外存活时间。     

钙通道拮抗剂体外对人精子运动参数的影响

目的　探讨钙通道拮抗剂在体外对人精子运动参数的影响。方法　将不同类型的钙通道拮抗剂在体外与生育男性经上游优化处理的精子分别共同孵育 10、2 0、3 0、60min ,与正常组进行对照研究。采用计算机辅助精子分析系统检测人精子运动参数 (精子活率、精子前向运动百分率、畸形率、平均路径速度、精子侧摆幅度和摆动幅度 )。结果　硝苯地平 (10 μmol/L)在体外对精子活率 (P <0 .0 1)、精子前向运动百分率 (P <0 .0 1)、精子平均路径速度 (P <0 .0 0 1)、精子侧摆幅度 (P <0 .0 1)和摆动幅度 (P <0 .0 1)等指标的差异有非常显著性。结论　人精子细胞中存在L 型电压依赖性钙通道 ,且L 型钙通道拮抗剂可导致男性不育。     

壬基酚和镉离子在体外对小鼠精子顶体反应的影响

目的:研究壬基酚和镉离子在体外对小鼠精子顶体反应(AR)的影响。方法:从小鼠的输精管获得精子,体外培养使精子获能,加30μmol/L的A23187诱导精子顶体反应,然后使用不同浓度的壬基酚(10、20、30、60、100μmol/L),或者镉离子(500、2500、5000μmol/L)处理,对照组使用相应的载体溶剂处理。用FITC-PSA荧光染色法分析精子顶体反应。结果:当壬基酚浓度<30μmol/L时,小鼠精子顶体反应率与对照组比较没有显著差异(P>0.05),而当壬基酚浓度>60μmol/L时能够显著地抑制小鼠精子顶体反应发生率(P<0.01),并且观察到精子存活率随着壬基酚浓度增加而降低。与壬基酚作用不同,用镉离子对小鼠精子进行处理,在所选浓度内(500~5 000μmol/L)均对精子顶体反应无显著影响(P>0.05),且精子存活率与镉离子浓度变化无关。结论:壬基酚与镉离子对小鼠精子发生的作用是通过不同的途径来实现的,前者可以直接抑制顶体反应,而后者则与精子顶体反应无关。     

人参皂甙Rg3对胰腺癌血管生成拟态的作用研究

目的探究人参皂甙Rg3对胰腺癌血管生成拟态作用。方法体外用鼠尾I型胶原蛋白建立三维培养体系,观察胰腺癌SW-1990、Panc-1、Bxpc-3、MiaPaCa-2四株细胞株形成血管生成拟态能力,应用PASR染色筛选出能够形成血管生成拟态的细胞株;用该株细胞建立体外模型,不同浓度人参皂甙Rg3（0、25、50、100、200μmol／L）处理模型,观察人参皂甙Rg3对该细胞株形成血管生成拟态的影响;进一步利用荧光定量PCR技术和Western blotting检测血管生成拟态相关指标MMP-2、MMP-9表达。结果胰腺癌SW-1990、Panc-1、Bxpc-3、MiaPaCa-2四株细胞株中,SW-1990在体外用鼠尾Ⅰ型胶原蛋白建立三维培养体系中培养72h后能够形成血管生成拟态。荧光PCR技术和Western blotting检测发现,与对照组相比,用药组MMP-2和MMP-9在mRNA水平和蛋白水平表达均下降：与空白组相比,各用药组MMP-2 mRNA水平和蛋白水平表达差别均有统计学意义（P〈0．05）;MMP-9在mRNA水平和蛋白水平表达情况,25μmol／L组与空白组比较无统计学意义（P〉0．05）,50μmol／L组、100μmol／L组、200μmol／L组与空白组比较差别有统计学意义（P〈0．05）。结论人参皂甙Rg3能够抑制胰腺癌血管生成拟态的形成,下调MMP-2和MMP-9的表达可能是其机制之一。     

POMHEX可抑制小鼠精子运动和获能

目的 探究抗肿瘤药物POMHEX对成熟小鼠精子功能的影响。方法 将小鼠精子在体外暴露于POMHEX(0.1～100μmol/L),通过计算机辅助精液分析系统评估精子活动率和前向运动精子百分率；伊红-苯胺黑染色法检测精子存活率；荧光素-荧光素酶系统检测精子ATP水平；蛋白质印迹实验检测精子获能相关蛋白质酪氨酸磷酸化；考马斯亮蓝G250染色检测精子顶体反应。结果 在不影响存活率的条件下，0.1μmol/L及以上浓度POMHEX可降低小鼠精子活动率(P<0.01)和前向运动精子百分率(P<0.05);1μmol/L及以上浓度可抑制小鼠精子ATP水平(P<0.01)和获能过程蛋白质酪氨酸磷酸化的增加。POMHEX虽然不影响小鼠精子的自发顶体反应，但在5μmol/L及以上浓度会抑制由钙离子载体A23187诱导的顶体反应(P<0.01)。结论 抗肿瘤药物POMHEX在体外可抑制小鼠精子运动和获能，对小鼠精子造成生殖毒性。提示接受POMHEX治疗的患者可提前进行生育力保存。     

Zeta potential of human X- and Y-bearing sperm

The zeta potential of human X- and Y-bearing sperm was measured by two different methods: (i) using an electrophoretic light scattering spectrophotometer, and (ii) using a laser-rotating prism. The X- and Y-bearing sperm were separated by free-flow electrophoresis, and their purities were determined by staining for the F-body using quinacrine mustard. The zeta potential of the sperm in the fraction containing more than 80% Y-bearing sperm was approximately -16 mV, whereas that of sperm in the fraction containing more than 95% X-bearing sperm was approximately -20 mV. In other words, the net negative-charge on the cell surface of human X-bearing sperm is higher than that of Y-bearing sperm.     

Role of the epididymis in sperm competition

Although it is generally understood that the testes recruited kidney ducts for reproductive function during the evolution of vertebrates, little is understood of the biological significance of the adaptation. In the context of the evolution of the mammalian epididymis, this report provides evidence that a major role of the epididymis is to enhance a male＇s chance of achieving paternity in a competitive mating system. A unique example of sperm cooperation in monotremes is used as evidence that the epididymis produces sperm competition proteins to form groups of 100 sperm into bundles that have a forward motility nearly thrice that of individual spermatozoa. As it required 3-h incubation in vitro under capacitation conditions to release motile sperm from the bundles, it is suggested that the monotremes provide an example of capacitation that is quite different from capacitation in higher mammals. It is suggested that variation between species in the intensity of sperm competition could explain the variation that occurs between species in the amount of post-testicular sperm maturation and storage in the epididymis, an explanation of why the human epididymis does not play as important a role in reproduction as the epididymis of most mammals. （Asian J Androl 2007 July; 9： 493-499）     

精子尾部卷曲试验及影响因素的探讨

本文介绍了精子尾部卷曲试验及影响因素（时间、温度、ｐＨ）。３０例正常生育男性和４３例不育男性的精液分析表明，精子尾部卷曲率与精子活率具有高度的相关性（ｒ＝０．９６２５），与精子密度无相关性（ｒ＝０．１６２）；男性生育组与不育组之间的精子尾部卷曲率具有显著性差异（ｘ±ｓ，百分率分别为７５．２１±２０．０１和５９．４３±２０．５４，Ｐ＜０．０５）。     

Activation of Spermatozoal Forward Migration in vitro by Hydrocortisone

The effects of hydrocortisone and cortisone on spermatozoal motility in vitro were tested. Hydrocortisone at concentrations of 50, 100 and 1000 nmoles/ml was effective in activating in vitro the forward migration and the motility of boar spermatozoa recovered from the cauda epididymidis. Where boar epididymal spermatozoa were incubated with hydrocortisone at concentrations of 50, 100 and 1000 nmoles/ml for between 0 and 24 h at 25°C in vitro , the spermatozoal motility was significantly higher than where no hydrocortisone was added. With ejaculated boar spermatozoa, hydrocortisone at concentrations of 100 and 1000 nmoles/ml increased the spermatozoal motility for between 0 and 2 h and at a concentration of 50 nmoles/ml increased spermatozoal motility for between 2 and 24 h at 25°C in vitro. After 4 h incubation, the effect of hydrocortisone at a concentration of 100 nmoles/ml on boar ejaculated sperm motility was not significantly different from the control. But, hydrocortisone at a concentration of 1000 nmoles/ml inhibited the forward migration of boar ejaculated sperm after it had been incubated with the sperm for 6 h. Cortisone, although structurally similar to hydrocortisone, had no significant effect in improving the motility of boar spermatozoa.
Both hydrocortisone and cortisone had no demonstrable effect on the forward migration and the motility of human spermatozoa in vitro. ,     

Characterisation of a subpopulation of sperm with massive nuclear damage,as recognised with the sperm chromatin dispersion test

Assessment of human sperm DNA fragmentation by the sperm chromatin dispersion (SCD) test is based on the detection of haloes of spreading DNA loops after sequential DNA denaturing and protamine removal. After the SCD test, sperm without DNA fragmentation show chromatin haloes emerging from the central nuclear core, while sperm containing fragmented DNA present small or no haloes. The nuclear degraded sperm are recognised as a differentiated category within the sperm with fragmented DNA, whose cores appear irregularly and/or faintly stained. This subpopulation is more prevalent in patients with varicocele. Protein staining with 2.7‐dibrom‐4‐hydroxy‐mercury‐fluorescein demonstrated that degraded sperm intensely lose nuclear core proteins after the SCD processing. Moreover, degraded sperm are 65% more faintly labelled for DNA breaks after in situ nick translation (ISNT) on average, due to extensive DNA loss. A two‐dimensional comet assay under sequential neutral and alkaline conditions demonstrated that degraded sperm contain both massive double‐ and single‐strand DNA breaks. The degraded sperm appear as a subpopulation with stronger nuclear damage, affecting both DNA and protein fractions, possibly due to intense intratesticular oxidative stress, what could explain its higher proportion in patients with varicocele.     

Changes in human sperm chromatin stability during preparation for in-vitro fertilization

This study was designed to define the effects of sperm preparation on sperm chromatin stability in relation to in-vitro fertilization (IVF). Semen samples used for IVF-embryo transfer (ET) in the treatment of infertility due to tubal factors were studied. Cases with semen variables below reference limits in previous samples were excluded. Sperm were prepared by a swim-up technique employing either of two different tissue culture media, Ham's F-10 or Earle's balanced salt solution. Sperm chromatin stability was tested by exposure both to sodium dodecyl sulphate (SDS) only and SDS together with a zinc-chelating agent, disodium ethylene diamine tetraacetate (SDS-EDTA). Sperm head swell scores were defined under different experimental conditions and the relationship to sperm motility, morphology, fertilization rate and pregnancy occurrence was tested. No differences were seen between the chromatin stability of sperm from the original sample and that after swim-up preparation, neither immediately after completion of the swim-up procedure, nor at the time of insemination of ova. With time, the chromatin became more stable, which occurred to a similar extent both in the original sample and in swim-up preparations using Ham's F-10. Otherwise, sperm chromatin stability was unaffected by either of the two media used for swim-up. At higher incubation temperatures, decondensation in SDS was enhanced. Altogether, no correlation was found between sperm chromatin stability or enhancement of decondensation by temperature and the success of IVF treatment expressed in fertilization rates or pregnancies. The results are reassuring in that only small changes in sperm chromatin stability occurred during the preparation for IVF. As long as semen of presumably good quality is used, these changes in chromatin stability do not seem to be of clinical importance.     

The effect of serum on motility of human spermatozoa in culture

The motility of spermatozoa was higher at 6-22 h in Ham's F10 culture medium supplemented with 20-30% human serum than with lower proportions of serum or with 1% human serum albumin. Heat treatment (56 degrees C, 1 h), charcoal extraction and dialysis (18,000 molecular weight cut-off) of the serum did not reduce sperm motility suggesting that high molecular weight components are responsible for maintenance of motility. Renewing the medium (Ham's F10 with 30% serum) at 7 h resulted in better sperm motility and velocity at 22 h. At 22 h the pO2, pCO2, pH and sodium concentrations were not different in replenished and control cultures, but the concentration of glucose was higher and that of potassium lower if the medium was changed. These results suggest that addition of 20-30% human serum and renewal of medium at intervals is beneficial for sperm culture and may be of use in in vitro fertilization.     

Two Plasminogen Activators, Streptokinase and Urokinase, Stimulate Human Sperm Motility

The stimulatory effects of two plasminogen activators, namely streptokinase and urokinase, were measured with a trans-membrane migration method. Both drugs induced maximal motility increase at a concentration of 200 international unit/ml; the amplitude of maximal motility increase ranged from 17% to 19% of control. Although their stimulatory effects were much less than those of calcium regulating agents, the clinical application of these two drugs for improving the successful rate of artificial insemination deserves further investigation because the action site is seminal plasma rather than sperm.     

Evaluation of different doses of mashua (Tropaeolum tuberosum) on the reduction of sperm production, motility and morphology in adult male rats

Mashua is an edible-tuber crop that grows in the Andean region. Folk medicine describes the use of mashua to reduce reproductive function in men. The present study aimed: (i) to determine whether different doses of mashua (0.01, 0.1, 1 and 2 g kg(-1)) produced a dose-response reduction on sperm production and quality; and, (ii) to determine whether these anti-reproductive effects of mashua can be reversible after cessation of treatment (12 and 24 days of recovery time). Mashua-treated rats showed lower values of daily sperm production, epididymal and vas deferens sperm count and sperm motility; meanwhile, mashua increased the percentage of abnormal sperm morphology and epididymal sperm transit rate. The following variables follow a dose-response effect: sperm number in vas deferens, sperm motility and sperm transit rate. In addition, it was demonstrated that the reduction in reproduction function in male rats treated with mashua was reversible after 24 days of recovery time. Finally, lower doses mashua reduces sperm number and quality (motility and morphology), and these adverse effects on male reproductive system may be reversible after 24 days after cessation of the treatment.     

ICSI中不同来源精子对临床结局的影响

目的:分析不同来源精子在卵细胞胞质内单精子注射(ICSI)后的临床结局。方法:将682个ICSI治疗周期分为:射出精子组(598例)、经皮附睾精子抽吸术(PESA)得到精子组(58例)、睾丸精子获取术(TSE)得到精子组(26例),比较三组的受精率、临床妊娠率、种植率、流产率、异位妊娠率以及分娩率之间的差别。结果:TSE组受精率明显低于射出精子组和PESA组(81.06%vs87.95%,87.82%,P<0.05);射出精子组、PESA组和TSE组的妊娠率(39.46%,48.28%,34.62%)、种植率(19.80%,23.80%,18.34%)、流产率(13.13%,17.86%,11.11%)、异位妊娠率(5.51%,7.14%,11.11%)、分娩率(32.11%,36.21%,26.92%),均没有显著差异(P>0.05)。结论:虽然TSE来源精子对ICSI受精率有所影响,但射出精子和手术(TSE和PESA)来源精子对妊娠结局没有影响。