人精子体外氧化损伤对线粒体tRNA~(LeuUUR)基因的影响

目的:探讨活性氧(ROS)对人类精子线粒体tRNALeuUUR基因的氧化损伤。方法:采用Percoll梯度离心法筛选具有正常生理功能的精子,作为正常精子模型,并分为损伤组20例和对照组20例,分别加入次黄嘌呤-黄嘌呤氧化酶体系或不予处理,37℃有氧环境中孵育60min。分别提取精子DNA,以Fpg酶切损伤碱基并采用接头介导PCR(LM-PCR)检测线粒体tRNALeuUUR基因的氧化损伤。采用Rhodamine(Rh123)荧光探针标记精子,通过流式细胞仪检测线粒体膜电位,观察精子的功能。结果:与对照组相比,损伤组精子孵育后线粒体膜电位明显降低[(116.27±11.72)%vs(64.00±4.88)%,P<0.05]。Fpg酶切和LM-PCR显示精子线粒体tRNALeuUUR基因损伤。结论:ROS可能通过对精子线粒体tRNALeuUUR基因氧化损伤而影响精子功能(线粒体膜电位明显降低),从而引起不育。     

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。     

白藜芦醇对人精子冷冻损伤的保护作用

目的:通过在人精子冷冻保护液中添加白藜芦醇,研究其对冻融后精子质量和功能的影响。方法:选择正常精液与少弱精子症样本各50例,液化后的精液样本分别与甘油-卵黄-柠檬酸盐(GEYC)冷冻保护液或含有30μmol/L白藜芦醇的GEYC冷冻保护液混匀。冷冻复苏前后,进行精子活力、存活率及顶体反应分析。采用丙二醛(MDA)及活性氧(ROS)检测试剂盒评估精子脂质过氧化程度及ROS水平。通过罗丹明123(Rh123)染色法及TUNEL试验检测精子线粒体膜电位及DNA损伤。结果:在正常精液和少弱精子症样本组中,与各组冷冻前新鲜精液相比,冻融后前向运动精子百分率、总活力、存活率、线粒体膜电位及顶体反应率均显著下降(P0.05),而精子ROS、MDA水平和DNA碎片指数(DFI)均显著升高(P0.05)。冷冻保护液中添加30μmol/L白藜芦醇后,正常精液组前向运动精子百分率[(43.1±6.3)%]、总活力[(56.9±7.4)%]、存活率[(67.5±5.6)%]、线粒体膜电位[(63.4±7.5)%]及顶体反应百分率[(26.3±4.7)%]较冷冻对照(未加白藜芦醇)的前向运动精子百分率[(32.7±4.8)%]、总活力[(44.8±6.9)%]、存活率[(52.3±6.1)%]、线粒体膜电位[(56.5±7.0)%]及顶体反应百分率[(16.6±3.8)%]均显著提高(P0.05),而精子ROS、MDA水平和DFI较冷冻对照均显著降低(P0.05)。在少弱精子症组中,添加白藜芦醇也均显著地提高了冷冻后精子前向运动百分率、总活力百分率、存活率、线粒体膜电位及顶体反应百分率,尤其是DFI[28.5±4.8)%]较冷冻对照[(36.3±5.7)%]显著降低(P0.01)。结论:在精液冷冻保护液中添加白藜芦醇可以通过降低精子内ROS水平减少精子冷冻损伤,从而改善解冻后精子质量和功能。     

活性氧致人精子运动功能和存活力变化的分析

目的 :对活性氧 (ROS)作用后精子的运动参数和存活率的变化进行分析 ,以证实ROS是否为引起精子运动功能障碍的病因之一。　方法 :采用Percoll梯度离心法选择具有正常生理功能的人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 ,在有氧环境下与精子模型孵育后 ,运用计算机辅助精液分析 (CASA)系统 ,分析ROS攻击后精子运动参数的改变。　结果 :与对照组相比 ,正常精子模型与ROS孵育 30min后 ,精子活动率、曲线速度 (VCL)、直线速度 (VSL)、平均路径速度 (VAP)均明显下降 (P <0 .0 0 1) ,头侧摆幅度 (ALH)尚无明显改变 (P >0 .0 5 ) ,而与ROS孵育 6 0min后 ,精子运动功能近乎丧失 ,精子主要运动参数趋向于零。　结论 :ROS与正常精子作用后 ,可导致精子运动功能下降 ,从而证实ROS为精子运动功能障碍的病因之一。     

龟龄集胶囊对弱精子症精子线粒体功能的作用机制研究

目的观察龟龄集胶囊对特发性弱精子症精子线粒体的作用,探讨龟龄集胶囊治疗特发性弱精子症的机制。方法根据特发性弱精子症诊断标准,筛选特发性弱精子症且中医辨证属肾阳虚的患者253例为治疗组,根据前向运动(A级+B级)精子的百分率分成3组:35%≤A组500/0、20%≤B组35%、C组20%;选取61名正常生育男性为空白对照组。A、B、c组均用龟龄集胶囊治疗,每次0.6 g,每日2次,4个月为一个疗程。对各组精子治疗前后均作精子线粒体功能检测和精液分析,并观察各组精子密度、精子活力、精子线粒体琥珀酸脱氢酶(SDt{)、线粒体膜电位(MMP)治疗前后的变化。结果 (1)各组治疗后线粒体功能SDH、MMP玫善,差异均有统计学意义(P0.01)。(2)各组前向运动精子活力均有明显提高(P0.01)。结论龟龄集胶囊能有效改善特发性弱精子症精子活力,其机制是改善精子线粒体琥珀酸脱氢酶和线粒体膜电位,从而提高了线粒体的功能。     

精子线粒体琥珀酸脱氢酶的检测及意义

目的 :采用一种简便、快速的检测精子线粒体琥珀酸脱氢酶 (SDH)的改良方法检测精子SDH ,并评价其与精子活动率及存活率的关系。　方法 :4 6例年龄为 2 5～ 36岁 (平均 31岁 )生育与不育男性精液标本 ,采用计算机辅助精液分析系统检测精子活动率 ,应用改良SDH检测法检测精子SDH ,通过死、活精子荧光分子探针染色检测精子存活率 ,分析精子活动率、存活率及精子SDH阳性率之间的关系。　结果 :4 6例生育与不育男性精子活动率为( 6 7.33± 7.37) % ,存活率为 ( 79.78± 7.6 5 ) % ,精子SDH阳性率为 ( 74 .74± 8.2 9) % ;精子活动率、存活率及精子SDH阳性率 3者之间均存在显著相关性 (P均 <0 .0 1)。　结论 :精子线粒体SDH检测对评价精子线粒体功能具有重要意义 ,还可作为精子存活率的辅助指标     

石萆汤通过调节线粒体膜蛋白PHB保护线粒体膜电位增强精子功能的探究

目的通过对90例男性不育弱精子症患者及30例正常男性进行分组处理观察石萆汤治疗弱精子症的可能靶点,探究石萆汤治疗肾虚夹湿型弱精子症的分子生物学机制。方法选择2016年8月～2018年6月在福建中医药大学附属人民医院男科门诊就诊的男性不育弱精子症患者和生殖体检者共120例,其中30例正常组与30例弱精子症组患者暂时未接受治疗,60例治疗组患者给予石萆汤治疗16周。治疗前后采用Western blot检测患者精子线粒体膜蛋白PHB及JC-1流式细胞仪检测线粒体膜电位。结果由Western blot结果,我们可以看到弱精子症组的患者精子线粒体膜蛋白PHB表达量明显低于正常组(P0.05);在给予石萆汤治疗16周后,治疗组患者精子线粒体膜蛋白PHB蛋白表达量显著高于弱精子症组(P0.05)。通过对3组患者精子JC-1染色后进行流式细胞仪检测发现弱精子症组患者精子线粒体膜电位(红色荧光散点)与正常组和治疗组相比偏向下移,提示弱精子组患者精子症线粒体膜电位降低。而经过石萆汤治疗16周后流式红色荧光散点上移差异具有统计学意义(P0.05)。结论石萆汤治疗肾虚夹湿型弱精子症可能是通过调控精子线粒体膜蛋白PHB表达进而改变精子线粒体膜电位,最终提高精子活力,增强精子功能,值得在今后临床中应用,同时线粒体膜蛋白PHB可对精子功能进行衡量,可在今后的临床检验中进行检测反映精子功能与状态。     

孕酮趋化诱导人精子线粒体功能与ROS产生的关系

目的:使用计算机辅助精液分析仪和流式细胞分析仪,比较经上游法处理前后、孕酮诱导上游法处理前后精子线粒体相对活性以及活性氧自由基(ROS)阳性精子比例的变化,研究受精精子的线粒体功能特点。方法:收集正常人类精子(达到《世界卫生组织人类精液检查与处理实验室手册》第5版标准),利用"上游法"获能培养1 h,获得"获能前组"和"获能后组"精子。获能后组的精子继续在37℃和5%二氧化碳培养箱中,使用"孕酮诱导"垂直上游法再处理1 h,获得孕酮诱导超激活运动精子,分别称为"孕酮诱导组"和"对照组"。分别使用线粒体膜电位特异性染色剂JC-1和ROS染色剂DCF,标记人类精子线粒体膜电位,并计算线粒体相对活性指数,标记ROS阳性细胞比例。结果:正常人类精子经获能处理及继续垂直上游后,线粒体相对活性(高活性线粒体/低活性线粒体比值)显著上升。获能前组、获能后组、对照组和孕酮诱导组的线粒体活性分别为1.42、6.23、14.36和12.33;获能前组、获能后组、对照组和孕酮诱导组含有均衡线粒体(含有等量高膜电位和低膜电位的线粒体)比例分别为21.64%、4.27%、5.03%和8.57%,说明孕酮诱导能够在激活线粒体活性的同时,维持低活性线粒体的比例。ROS同时在精子的头、颈、尾部出现,获能前组、获能后组、对照组和孕酮诱导组ROS阳性细胞比例分别为2.89%、0.7%,4.25%和1.90%。上游1 h后的精子ROS阳性比例显著低于上游前精子。再经过1 h垂直上游后,"对照组"精子的ROS阳性比例大大增加;孕酮诱导组精子的ROS阳性比例显著低于"对照组"。结论:孕酮体外诱导上游精子,能够显著提高精子运动速度,使其呈现超激活运动状态,同时抑制精子的线粒体相对活性持续增高,保持线粒体活性均衡,降低ROS产量,可能有利于体外受精率和胚胎质量的提高。     

尿激酶型纤溶酶原激活因子对小鼠获能精子线粒体膜电位的影响

目的:检测尿激酶型纤溶酶原激活因子(uPA)体外对小鼠获能精子线粒体膜电位的影响。方法:采用线粒体膜电位荧光染料JC-1,利用流式细胞仪和荧光显微镜检测分别与uPA(实验组)和BWW液(对照组)共孵育0、5、15、30、60min的小鼠获能精子线粒体膜电位状态。结果:①在uPA作用第5、15min时,精子体内平均荧光强度较作用前显著增加,高膜电位的精子数量百分率相应增加(P<0.05)。②与对照组相比,实验组在uPA作用5、15min时的高膜电位精子数量百分率,以及作用15、30、60min时的精子线粒体膜电位平均荧光强度显著增加(P<0.05)。结论:uPA在体外可以增加小鼠获能精子的线粒体膜电位,并使高膜电位状态维持一段时间,为获能精子提供充足的能量供应。     

高、低生育力捐精者复苏精液功能指标的比较性研究(附40例报告)

目的:探讨可以全面有效评估捐精者生育力的精子功能指标,运用于复苏精液标本的筛选,旨在提高辅助生殖技术成功率。方法:根据捐精者精液使用的妊娠结局,收集上海市人类精子库高、低生育力捐精者的冷冻精液标本各20例,比较两组精液标本复苏后的精子浓度、活力、正常形态率、顶体完整率、DNA完整性以及线粒体膜电位。结果:高、低生育力组精液复苏经系列评估后,正常形态率分别为(18.50±6.10)%、(14.42±6.44)%;顶体完整率分别为(86.17±4.49)%、(80.04±7.52)%;精子DNA碎片率分别为(9.21±3.22)%、(15.72±8.20)%,以上指标经统计学分析两组之间具有显著性差异(P0.05)。但线粒体膜电位高生育力组[(56.75±18.80)%]与低生育力组[(52.23±18.86)%]之间无显著性差异(P0.05)。精子线粒体膜电位与精子活力呈显著正相关(r=0.760,P0.05),其他功能指标与精子浓度、活力无显著相关。结论:精子浓度、活力与正常形态率、顶体完整率以及DNA完整性可以有效评估捐精者复苏精液生育能力。     

Protection of melatonin against damage of sperm mito-chondrial function induced by reactive oxygen species

Aim: To study the mitochondrial function damage of sperm in-duced by reactive oxygen species (ROS) and the protection of melatonin (MLT) against the damage. Methods: Normal function spermatozoa were selected from semen samples by Percoll gradi-ent centrifugation technique. The ROS generated by the hypoxan-thine xanthine oxidase system was incubated with the normal sper-matozoa in the presence or absence of MLT (6 retool/L) for 30 and 60 minutes.     

Effects of reactive oxygen species from activated leucocytes on human sperm motility, viability and morphology

The accumulated data suggest that inflammation can increase the level of reactive oxygen species (ROS), which contribute to impaired sperm function and male infertility. Therefore, we propose that inflammation-mediated production of ROS in male and female reproductive tracts hinder sperm fertilisation. To test this hypothesis, phorbol myristate acetate (PMA) with polymorphonuclear leucocytes (PMNs) was applied to generate endogenous ROS. We evaluated the time-dependent effects of ROS on human sperm motility, viability and mitochondrial membrane potential (MMP). The results showed that after treatment with PMA and PMNs, the motility of human spermatozoa significantly decreased to 50% on Day 1 and 15% on Day 4 compared with that of the, respectively, negative controls (P = 0.012). The viability of human spermatozoa decreased on Day 4 of PMA + PMNs treatment (P = 0.028). The MMP of human spermatozoa significantly decreased from Day 2 to Day 4 in the PMA + PMN group compared with that of the controls (P = 0.019). Taken together, the 4-day cultivation approach provided an accurate evaluation of sperm quality, especially sperm motility and MMP. Our findings indicated that endogenous inflammation increased ROS levels, which might induce sperm oxidative damage. Additionally, sperm motility might be one of the earliest and most sensitive indicators of this damage.     

Effects of semen processing on the generation of reactive oxygen species and mitochondrial membrane potential of human spermatozoa

This study was designed to evaluate the effects of semen processing on the generation of intracellular reactive oxygen species (ROS) and mitochondrial membrane potential (MMP) in spermatozoa, and to develop reliable indexes for the evaluation of sperm quality during sperm preparation. Swim-up and density gradient centrifugation methods were used to separate semen in oligoasthenoteratozoospermia (OAT), leucocytospermia (LC) and normozoospermia groups. Levels of ROS and MMP were measured by flow cytometry. Before preparation, the patients with abnormal semen parameters had a lower MMP and higher ROS, and there was a negative correlation between MMP and ROS. The levels of MMP and ROS increased significantly, especially ROS produced by swim-up. A significant difference was found between the correlation of MMP and total normal motile sperm count after preparation in the OAT group. The level of ROS was associated with the amount of white blood cells in the LC group. The MMP can be used as an objective index to evaluate the sperm quality of OAT patients, and the combination of MMP and ROS can be used to assess the efficiency of sperm preparation in LC patients. These findings can guide selection of the ideal sperm separation technique for different sperm samples.     

Effect of mitochondrial calcium uniporter blocking on human spermatozoa

Calcium (Ca²⁺) regulates a number of essential processes in spermatozoa. Ca²⁺ is taken up by mitochondria via the mitochondrial calcium uniporter (mCU). Oxygen‐bridged dinuclear ruthenium amine complex (Ru360) has been used to study mCU because it is a potent and specific inhibitor of this channel. In bovine spermatozoa, it has been demonstrated that mitochondrial calcium uptake inhibition adversely affects the capacitation process. It has been demonstrated in human spermatozoa that mCU blocking, through Ru360, prevents apoptosis; however, the contribution of the mCU to normal human sperm function has not been studied. Therefore, the aim of this study was to evaluate the effect of mCU blocking on human sperm function. Spermatozoa obtained from apparently healthy donors were incubated with 5 and 10 μm Ru360 for 4 h at 37 °C. Viability was assessed using propidium iodide staining; motility was determined by computer‐aided sperm analysis, adenosine triphosphate (ATP) levels using a luminescence‐based method, mitochondrial membrane potential (ΔΨm) using JC‐1 staining and reactive oxygen species (ROS) production using dihydroethidium dye. Our results show that mCU blocking significantly reduced total sperm motility and ATP levels without affecting sperm viability, ΔΨm and ROS production. In conclusion, mCU contributes to the maintenance of sperm motility and ATP levels in human spermatozoa.     

The effect of cysteine and glutamine on human sperm functional parameters during vitrification

Assuming the adverse effects of reactive oxygen species (ROS) on sperm function, this study was conducted to assess the effects of cysteine and glutamine as effective antioxidants on human sperm parameters under vitrification. Twenty normozoospermic samples were used. The samples were subjected to a vitrification process and cysteine (5 and 10 mM) and glutamine (10 and 15 mM). The sperm motility parameters, mitochondrial membrane potential (MMP), plasma membrane integrity (PMI), DNA damage and intracellular ROS damage were assessed for each sample. Statistical analyses showed that motility, mitochondrial membrane potential and DNA damage decreased in the vitrified groups with cysteine 5, 10 mM and glutamine 10, 15 mM separately. Also intracellular ROS increased significantly compared to the fresh group (p < .05). No significant differences were observed for PMI compared with the fresh group (p > .05). Supplementation of cysteine and glutamine in both concentrations separately decreased intracellular ROS and DNA damage of spermatozoa with significant increase in PMI, MMP and progressive motility compared to vitrified control group (p < .05). The results showed no significant effect of a specific concentration in cysteine and glutamine on sperm parameters compared to other concentrations. Both amino acids have the potential to improve the harmful effects of freezing on sperm parameters.     

Antioxidant effects of penicillamine against in vitro-induced oxidative stress in human spermatozoa

Oxidative stress contributes importantly to the aetiology of male infertility, impairing sperm function. The protective effect of antioxidants on seminal parameters has been established, and the antioxidant penicillamine has shown beneficial effects; however, its protective effect on human spermatozoa exposed to oxidative stress has not been reported. The objective of this work was to evaluate the effect of penicillamine on human spermatozoa exposed in vitro to oxidative stress. First, the effect of penicillamine on spermatozoa from normozoospermic donors was evaluated. Then, the effect of penicillamine on spermatozoa exposed to oxidative stress induced separately by ionomycin and hydrogen peroxide (H₂O₂) was analysed. An untreated control and a control treated only with the oxidative stress inducer were included. Reactive oxygen species (ROS) levels, viability, mitochondrial membrane potential (MMP) and motility were analysed. The results showed that penicillamine, added to the incubation medium, decreased the ROS levels induced by ionomycin and H₂O₂, and this effect was associated with better preservation of MMP, motility, and ATP levels. These results highlight the potential advantages of penicillamine supplementation of sperm culture medium, especially for semen samples with high ROS levels and also in circumstances where laboratory handling can cause an increase in ROS production.     

The effect of reactive oxygen species on equine sperm motility, viability, acrosomal integrity, mitochondrial membrane potential, and membrane lipid peroxidation

The objective of this study was to examine the influence of reactive oxygen species (ROS), generated through the use of the xanthine (X)-xanthine oxidase (XO) system, on equine sperm motility, viability, acrosomal integrity, mitochondrial membrane potential, and membrane lipid peroxidation. Equine spermatozoa were separated from seminal plasma on a discontinuous Percoll gradient, and spermatozoa were incubated with 0.6 mM X and 0.05 U/mL XO for 30 minutes. Catalase (150 U/mL), superoxide dismutase (SOD, 150 U/mL), or glutathione (GSH, 1.5 mM) were evaluated for their ability to preserve sperm function in the presence of the induced oxidative stress. At the end of the 30-minute incubation, sperm motility was determined by computer-assisted semen analysis. Viability and acrosomal integrity were determined by Hoechst-Pisum sativum staining, and mitochondrial membrane potential was determined by staining with JC-1. Incubation with the X-XO system led to a significant (P < .01) increase in hydrogen peroxide production and an associated decrease (P < .01) in motility parameters. Total motility was significantly (P < .01) lower in the presence of X-XO compared with the case of the control (29%+/-9% vs 73%+/-1%, respectively). Catalase, but not SOD, prevented a decline in motility secondary to oxidative stress (71%+/-4% vs 30%+/-3%, respectively). The addition of glutathione had an intermediate effect in preserving sperm motility at the end of the 30-minute incubation (53%+/-3%). No influence of X-XO could be determined on viability, acrosomal integrity, or mitochondrial membrane potential. In order to promote lipid peroxidation, samples were incubated with ferrous sulfate (0.64 mM) and sodium ascorbate (20 mM) for 2 hours after the X-XO incubation. No increase in membrane lipid peroxidation was detected. This study indicates that hydrogen peroxide is the major ROS responsible for damage to equine spermatozoa. The decrease in sperm motility associated with ROS occurs in the absence of any detectable decrease in viability, acrosomal integrity, or mitochondrial membrane potential or of any detectable increase in lipid peroxidation.