精子线粒体DNA与男性不育研究进展

精子线粒体DNA(mtDNA)与男性生育能力关系密切。mtDNA是裸露的DNA,缺少组蛋白和DNA结合蛋白的保护,易受活性氧(ROS)的攻击,造成氧化损伤,且mtDNA缺少有效的修复系统,ROS引起的精子mtDNA改变将破坏mtDNA编码蛋白的合成,影响线粒体的功能,这可能是男性不育重要遗传学因素。找到引起精子mtDNA损伤的原因及其与男性不育的关系是研究的重点。本文综述了精子mtDNA与男性不育关系的最新研究进展。     

精子线粒体DNA与男性不育

线粒体DNA是细胞质中独立的细胞器，是真核生物细胞内能量转换体系和供能中心。它含有一个独立的半自主复制的DNA，称为线粒体DNA(mitochondrial DNA，mtDNA)。1981年，完整的人mtDNA序列由Anderson等人测定。它是一个16569bp的双链闭合环状分子，编码13种蛋白质，22种tRNA和2种rRNA。这13种蛋白质都是呼吸链酶复合物的亚单位，与细胞核DNA编码生成的多肽一起参与氧化磷酸化(oxidative phosphorylation，OXPHOS)。细胞通过OXPHOS产生的三磷酸腺苷(adenosine triphosphate，ATP)占细胞生命活动所需能量的90％以上，因此mtDNA对维持细胞的正常功能起重要作用。     

精子密度与精子活动度的关联性研究

目的：探讨精子密度和精子活动度关系.方法：回顾性分析2005-2007年5 519份精液数据.患者年龄（32±5）岁,精液体积2～6 ml,精子密度（48.09±40.33）× 10^6/ml,a级精子（17.95±11.86）%,b级精子（14.64±7.80）%,c级精子（16.86±6.96）%,d级精子（48.38±19.03）%.检测方法：采用CASA系统对精液进行检测.SPSS13.0对于精子密度及精子活动度进行统计学分析.结果：年龄与精子密度之间不存在相关性.精子密度与各级精子活动度之间几乎均存在相关性关系,精子密度与精子活动度a、b、d间相关性低,与活动度c高度相关.以精子密度为自变量,精子活动度c为因变量,随精子密度的增加,活动度c呈上升趋势,两者间存在线性关系.结论：精子密度与活动度c存在关联性,推测精子发生与精子成熟调控机制存在共同的调控因素.     

Chk1/2基因表达对精子浓度和活力的影响

目的:研究精子中细胞周期检测点激酶1/2(Chk1和Chk2)基因的表达对精子浓度及活力的影响。方法:将精液样本根据精子浓度和活力(前向运动精子百分率)分为正常对照组、少精子症组、弱精子症组和少弱精子症组,每组20例。分别检测各组精子DNA碎片指数(DFI)、精子存活率,采用RT-PCR和Western印迹方法分别检测各组精子Chk1、Chk2的表达。结果:①4组DFI分别为21.24±6.93、19.67±7.64、21.52±6.92、19.28±11.55,无显著差异(P0.05);4组精子存活率分别为(83.48±9.87)%、(63.86±9.16)%、(50.45±16.99)%、(39.21±15.74%),与正常对照组相比,其余3组均有显著下降(P均0.05);②DFI30%和DFI≤30%两组精子的浓度、活力和精子存活率之间的差异有统计学意义(P0.01);③ RT-PCR检测结果显示,4组Chk1 mRNA相对表达量分别为0.73±0.22、0.62±0.14、1.03±0.39、0.92±0.071,各组间比较差异有显著性(P0.01),其与精子浓度呈正相关(b=80.661,P0.01),与精子活力呈负相关(b=-19.275,P0.01);4组Chk2mRNA相对表达量分别为0.66±0.30、0.27±0.09、0.59±0.19、0.42±0.11,各组间比较差异有显著性(P0.01),其与精子浓度呈负相关(b=-90.809,P0.01),与精子活力呈正相关(b=27.507,P0.01)。④Western印迹结果显示,4组Chk1蛋白相对表达量分别为0.63±0.05、0.42±0.03、1.13±0.08、0.87±0.07,各组间比较差异有显著性(P0.01),其与精子浓度呈正相关(b=55.74,P0.01),与精子活力呈负相关(b=-22.649,P0.01);4组Chk2蛋白相对表达量分别为1.23±0.36、0.37±0.16、0.87±0.08、0.68±0.12,各组间比较差异有显著性(P0.01),其与精子浓度呈负相关(b=-53.001,P0.01),与精子活力呈正相关(b=16.676,P0.01)。结论:Chk1和Chk2在人类精子中均有显著表达,在精子DNA出现损伤后,Chk1表达的增强可能促进了精子的凋亡导致弱精子症,而Chk2表达的增强则可能抑制了精子的生成而导致少精子症。     

精子线粒体膜电位评估男性生育力的作用研究进展

全球目前约有15％的适龄夫妇无法生育,其病因中男方因素占约20％,男方因素是导致适龄夫妇无法生育的重要原因之一[1,2].另外目前国内外最新流行病学显示男性精液质量呈逐年下降趋势[3].而不明原因的少、弱、畸形精子症,即特发性少、弱、畸形精子症,又占据男性不育中的较大比例,因此精液质量与男性生育力密切相关[45].目前...     

JC-1单标法流式细胞术检测精子线粒体膜电位的研究

目的:探讨应用荧光染料JC-1单色标记法进行流式细胞术检测精子线粒体膜电位(MMP)的可行性及其临床意义。方法:收集63例男性精液标本,分为生育组(n=31)和不育组(n=32)。通过计算机辅助精液分析系统进行精液常规分析。精液标本洗涤处理后用JC-1染色后上流式细胞仪分析,用发橙红色荧光精子百分率(JC-1+%)表示MMP正常精子的比例。结果:生育组精子JC-1+%为(75.89±15.69)%,显著高于不育组[(54.04±22.21)%,P=0.000]。63例标本中,JC-1+%与精子活动率呈显著正相关(r=0.610,P=0.000),与(a+b)级精子百分率呈显著正相关(r=0.614,P=0.000),与d级精子百分率呈显著负相关(r=-0.504,P=0.000)。JC-1+%与已建立的罗丹明/碘化吡啶双染法检测结果(Rh123+/PI-%)呈显著正相关(r=0.938,P=0.000)。结论:应用流式细胞术JC-1单标法检测精子MMP具有可行性,精子JC-1+%可作为男性不育的辅助诊断指标。     

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。     

精子线粒体功能障碍在弱精子症中的研究进展

世界范围内大约有15%的夫妇受不育症困扰,其中男性不育者的精液异常多表现为精子活力低下。研究表明,精子中线粒体膜电位、活性氧、线粒体内Ca2+含量、线粒体酶以及蛋白组活性、线粒体超微结构、mtDNA等的异常可引起精子活力下降。本文通过回顾线粒体与弱精子症的相关文献,阐述精子线粒体功能障碍在弱精子症中可能的作用,为弱精子症发生发展机制的研究提供新的思路。     

JC-1单标法检测精索静脉曲张患者精子线粒体膜电位的研究

目的:检测精索静脉曲张患者精子线粒体膜电位并探讨其临床意义。方法:将67例精索静脉曲张患者分为VC1组(精索静脉曲张1度,n=26)、VC2组(精索静脉曲张2度,n=21)和VC3组(精索静脉曲张3度,n=20),以正常生育男性为正常对照组(n=29)。通过计算机辅助精液分析系统进行精液常规分析。精液标本洗涤处理后用荧光染料JC-1染色后上流式细胞仪分析,检测精子线粒体膜电位(JC-1+%)。结果:VC1、VC2、VC3组精子线粒体膜电位[(56.29±16.32)%,P<0.05;(45.04±13.21)%,P<0.01;(31.63±12.91)%,P<0.01]均显著低于正常对照组[(76.21±13.96)%]。96例标本中,JC-1+%与(a+b)级精子百分率呈显著正相关(r=0.693,P=0.000)。结论:精索静脉曲张可引起精子线粒体膜电位降低,可能是导致男性不育的重要原因之一。     

Relationship between sperm mitochondrial membrane potential,sperm motility,and fertility potential

AIM:To analyze the relationship between sperm mitochondrial membrane potential and sperm motility parameters by means of a computer-assisted sperm analyzer (CASA) and in-vitro fertilization rate(%FR). METHODS: Semen samples were obtained from 26 men undergoing in vitro fertilization-embryo transfer (IVF-ET). Informed consent was obtained from all men prior to the study. Samples were prepared using wash and swim-up method in HEPES-HTF medium. The sperm motility (%MOT), progressive motility (%PMOT), average path velocity (VAP) microm/s), straight line velocity (VSL) (micro m/s), curvilinear velocity (VCL) (microm/s) and %hyperactivated sperm (%HA), and the %FR were assessed. The samples were incubated in the presence of 2.0 mciromol/L of 5,5',6,6'-tetra-chloro-1,1',3,3'-tetraethylbenzimidazolyl-carbocyanine iodide (JC-1) for 30 min at 37 degrees C in air and washed in PBS before flow cytometry (FACSCalibur: Becton Dickinson) analysis. The mitochondrial probe JC-1 was used to identify the mitochondrial membrane potential. The sperm was divided into three populations according to the fluorescence pattern as follows: the high mitochondrial membrane potential group (n=8), the moderate group (n=5), and the low group (n=13). Statistical analysis was performed using unpaired t-test. RESULTS:Significant differences were found between the high and the low groups in %MOT (91.1+/-8.5 vs 63.0+/-32.7, mean+/-SD), VAP (73.0+/-14.2 vs 52.1+/-12.5), VCL (127.0+/-28.1 vs 87.0+/-22.6), %HA (27.3+/-23.6 vs 7.2+/-9.0) and %FR [73.2 (48/56) vs 59.0 (69/117)]. No significant differences were found in other CASA parameters. CONCLUSION: When the sperm mitochondrial membrane potential increases, sperm motility parameters and fertility potential will also increase. The JC-1 dye method is useful to predict sperm fertility potential.     

The relationship between ejaculated ram sperm mitochondrial protein synthesis and motility

Summary. D-chloramphenicol, at concentrations of 20 and 40 μg/ml, inhibited over 80% of the newly synthesized mitochondrial proteins expressed by incorporation of ³H-amino acid mixture. At concentrations of 20 and 40 μgml⁻¹, D-chloramphenicol enhanced the collective motility of washed ram spermatozoa. The collective motility measured by the multichannel Reflectospermiograph system, significantly enhanced the motility wave frequencies and amplitudes by 24–17% and 65–32%, respectively. Furthermore, the longevity of the collective motility was prolonged by 12–19%. In about 20% of the cases, when the original sperm motility was low, it was found that 40 μg ml⁻¹ D-chloramphenicol has maximum stimulation effect on sperm motility in inverse fashion. Since the mitochondria are located adjacent to the motility initiation area, it can be speculated that the mitochondrial protein(s) directly inhibiting the axonemal-ATPase activity or indirectly blocking sperm metabolite, are essential for maintaining sperm motility.     

Relationship between sperm motility assessed with the Hamilton-Thorn motility analyzer and fertilization rates in vitro

To determine which sperm movement characteristics are related to in vitro fertilization rates, semen and swim-up preparations used for in vitro fertilization in 108 patients were assessed using the Hamilton-Thorn HTM-2030 Motility Analyzer (HTMA) and other sperm tests. There were highly significant correlations between manual and HTMA results for sperm concentration (Spearman r = 0.881; P less than 0.001) and the percentage of motile spermatozoa (Spearman r = 0.580; P less than 0.001). The percentage of motile spermatozoa with average path velocities greater than 10 microns/s and greater than 20 microns/s, straight line and curvilinear velocity, linearity (straight line velocity vs curvilinear velocity), amplitude of lateral head displacement, and beat-cross frequency were significantly higher in the insemination medium after selection of motile spermatozoa by the swim-up technique than in the semen. Linearity (P less than 0.01), the percentage of morphologically normal spermatozoa (P less than 0.05) and straight line velocity (P less than 0.05) in semen, and the percentage of motile spermatozoa with average path velocities greater than 10 microns/s in both semen (P less than 0.05) and insemination medium (P less than 0.05) were significantly correlated with in vitro fertilization rate when examined by a nonparametric (Spearman) test. With logistic regression analysis of all data, only the diagnoses of male infertility and tubal disease, linearity in semen, and the percentage of motile spermatozoa with average path velocities between 10 and 20 microns/s in insemination medium were significantly related to in vitro fertilization rates.(ABSTRACT TRUNCATED AT 250 WORDS)     

不育男性精子密度、活动率、活力与顶体酶活性关系的研究

目的探讨不育男性精子密度、活动率、活力、与精子顶体酶活性的关系.方法采用分光光度比色法测定精子顶体酶活性,并将其与精子密度、活动率和活力进行统计分析.结果随着精子密度、活动率、活力降低,精子顶体酶活性均明显降低(各组间比较均P＜0.001).结论不育男性精子顶体酶活性降低可影响精子密度、活动率和a, b级活力精子率,精子顶体酶活性可作为男性不育精子质量检测的指标之一.     

Sperm mitochondrial mutations as a cause of low sperm motility

We report the unique case of a 28-year-old man who, in spite of having a varicocele and a sperm concentration of 5 million/mL, of which 10% were motile and 20% had normal forms (oligoasthenoteratozoospermia [OAT]), was fertile. This was confirmed by paternity testing using 16 autosomal and 6 Y-chromosomal short tandem repeat (STR) loci. An analysis of mitochondrial genes that included cytochrome oxidase I (COI), cytochrome oxidase II (COII), adenosine triphosphate synthase6 (ATPase6), ATPase8, transfer ribonucleic acid (tRNA) serine I, tRNA lysine, and NADH dehydrogenase3 (ND3) revealed, for the first time, 9 missense and 27 silent mutations in the sperm's mitochondrial DNA (mtDNA) but not in the DNA from the blood cells. There was a 2-nucleotide deletion in the mitochondrial COII genes, introducing a stop codon, which might be responsible for low sperm motility.     

精子DNA冷冻损伤易感性与精子活动力相关性

目的研究不同活动力精子其核DNA对冷冻损伤的易感性。方法选取来本中心行精液常规分析,精子浓度5×106/ml、精子正常形态率4%者63例为研究对象。精子冷冻前后均采用染色质扩散(SCD)实验分析核DNA完整性,分别比较高活力组(d级30%,n=28)及低活力组(d级≥30%,n=35)精子在冷冻前后DNA完整性的差异。结果精液标本(n=63)经冷冻后,精子DNA碎片指数(SDF)较冷冻前显著升高[(23.1±12.5)%vs.(19.9±11.6)%,P0.05],其中代表精子DNA完整性较好的大晕轮精子比例显著降低[(71.9±13.3)%vs.(75.8±12.2)%,P0.05],而小晕轮精子比例则显著升高[(10.2±5.7)%vs.(8.2±3.9)%,P0.05]。在低活力组,精子SDF及大晕轮、小晕轮精子比例在冷冻前后亦差异显著(P0.05);而在高活力组,冷冻后仅小晕轮精子比例较冷冻前显著升高[(8.3±3.4)%vs.(6.8±3.0)%,P0.05],SDF及大晕轮、中晕轮、无晕轮的精子比例在冷冻前后均无显著差异(P0.05)。结论相比高活力组精子,低活力组精子核DNA冷冻耐受力较差,更易受损。     

Human sperm mitochondrial function related to motility: a flow and image cytometric assessment

Current evaluation of male fertility, routinely estimated by sperm count, motility, and morphology, provides only crude information about the fertility state of individuals. Both flow and image cytometry were applied to mitochondrial activity and sperm motility respectively. Sperm samples from fertile donors were concomitantly measured for Rhodamine 123 (Rh123) uptake (an estimation of mitochondrial activity), percentage of dead cells, and motility characteristics, such as percentage of motility, curvilinear velocity, and amplitude of lateral head displacement. These measurements were done under experimental conditions known to modulate sperm motility (temperature and time course survival in a capacitating medium). Bimodal distributions were found for Rh123 uptake. Flow cytometry-derived parameters were essentially time-dependent whereas motility characteristics were primarily temperature-dependent. Correlations were found between various flow cytometry-derived parameters and motility characteristics. Most of the correlations were obtained after a 24 h incubation in a capacitating medium. The most significant correlation in every experimental condition concerned the percentage of motile spermatozoa and the Rh123 uptakes. The drop in motility observed after a 24 h incubation was paralleled by a markedly lower drop in mitochondrial activity. The data suggest that these two complementary techniques represent an improvement in basic and/or clinical assessment of the functional spermatozoa status.     

Computerized sperm motility and application of sperm cryopreservation

An objective method for measuring sperm motion characteristics was developed on an Intellect 100 Quantel Image Analysis System suitable for various image cytometric applications. It provided overall analysis of percent motility (% MS) as well as individual and mean measurements of motion characteristics, including vigor characteristics such as curvilinear velocity (Vc), straight line velocity (Vsl), and trajectory pattern characteristics, that is, progressiveness ratio (PR) and amplitude of lateral head displacement (Alh). Evaluation of the method for reproducibility and accuracy showed reliable measurements of these parameters measured on a minimum of 70 motile sperm, sufficient to describe adequately the sperm population. A study was performed comparing motion characteristics of 30 semen samples falling in a normal range before and after cryopreservation in cryoprotector medium (CM). A mean motility rate of recovery (MRR) of 45% was obtained. Only sperm count and concentration in motile forms among initial semen variables correlated weakly with MRR. Velocity recovery rate (VRR) approached 1 with a marked variability among ejaculates. Distribution profile of Vc was highly modified by freezing in CM: spermatozoa that were initially fast and progressive were the most resistant to cryoaggression. PR and Alh values were little affected by freezing in CM. The tolerance of various samples from a given patient was highly variable for % MS and Alh and less variable for Vc and PR. This illustrates the difficulty in predicting the effect of freezing on motility characteristics and, therefore, of extrapolating from semen variables the ability of frozen-thawed samples to fertilize.     

Correlation between in vitro fertilization and human sperm density and motility

The conventional sperm characteristics of density (millions per milliliter) and motility, scored in a semi-subjective way, were correlated with results of an on-going in vitro fertilization and embryo transfer program. No male infertility patients were included in this study. Individual characteristics of the "successful" ejaculates are described. Sperm densities in the original ejaculate of more than 10 X 10(6) spermatozoa/ml did not significantly improve outcome (P less than 0.01). In contrast, sperm motility seemed to play the most important role, since most pregnancies (12/14) occurred using sperm samples with greater than or equal to 60% total motility (P less than 0.001). The incidence of multipronuclear fertilization is also described and discussed. These data, which were collected during 1984 in the in vitro fertilization unit of Professor R. Schoysman and coworkers (Vilvoorde, Brussels), may help to make fertilization in vitro and embryo transfer a viable method in cases of mild male subfertility, and to provide guidance in preparing some couples for the combined use of husband and donor semen if a sufficient number of oocytes are obtained.     

特发性弱精子症患者血清及精浆瘦素水平与精子活动力的关系

目的 通过研究特发性弱精子症(idiopathic asthenospermia,IAS)患者以及精液参数正常人群的血清、精浆瘦素,探讨瘦素与精子运动能力的关系.方法 IAS患者54例及精液参数正常者30例作为对照.常规CASA精液分析,放射免疫法检测血清及精浆瘦素.结果 排除体重指数差异的影响,(IAS患者与精液参数正常者体重指数比较差异无统计学意义,P>0.05),IAS患者血清瘦素水平与正常对照差异无统计学意义(P>0.05),而IAS患者精浆瘦素显著高于正常对照(P<0.05);精子活动率及精子活力与血清瘦素水平之间均无显著相关性(r=-0.213,P=0.249及r=-0.167,P=0.154),IAS患者的精子活动率及活力与精浆瘦素水平显著负相关(r=-0.31,P=0.034及r=-0.47,P=0.025).结论 IAS患者精浆瘦素水平显著增高,可能对精子运动能力有调控作用.