冷冻保存对正常和不育症精子PH-20表达和凋亡的影响

目的探讨冷冻保存对人精子膜蛋白PH-20表达和精子凋亡的影响。方法 14例正常生育力精液标本(A组)和20例不育症精液标本(B组)行冷冻保存。Western blotting检测PH-20蛋白在人精子中的表达,免疫荧光用来观察PH-20蛋白在人精子上的定位,应用末端脱氧核苷酸转移酶(Td T)介导的原位末端标记(TUNEL)法检测精子凋亡情况。结果解冻后正常生育组和不育组的PH-20/β-actin平均吸光度与冷冻前比较均有显著性下降(P0.05);解冻后正常生育组和不育组的PH-20阳性率与冷冻前比较均有显著性下降(P0.05)。解冻后正常生育组的精子凋亡率与冷冻前的比较差异无显著性意义(P0.05)。而解冻后不育组的精子凋亡率与冷冻前的比较均显著性下降(P0.05),且不育组的降低程度大于正常生育组。结论冷冻-解冻过程可引起精子PH-20蛋白表达减少和精子PH-20阳性率降低,但冷冻保存对正常生育者的精子凋亡率无显著影响。     

人精子膜蛋白P34H表达与透明质酸酶活性的关系

目的探讨人精子膜蛋白P34H表达和顶体内透明质酸酶活性的关系。方法收集88例精液标本,其中正常生育组20例,不育男性68例。参照世界卫生组织(WHO)标准对标本进行精液常规分析,根据精液参数的不同,将68例不育标本分为正常精液参数不育组和精液参数异常不育组。Western blotting检测P34H蛋白在人精子中的表达,免疫荧光观察P34H蛋白在精子表面的阳性表达率,采用改良透明质酸钠-明胶底物膜法检测精子顶体内透明质酸酶(HYD)活性(HYD阳性反应率、HYD活性强度)。结果正常精液参数不育组和精液参数异常不育组的精子P34H/β-actin平均吸光度、精子P34H标记阳性率均明显低于正常生育组,经统计学分析差异均有显著性(P0.05);正常精液参数不育组和精液参数异常不育组的HYD活性(HYD阳性反应率、HYD活性强度)与正常生育组比较均有显著下降(P0.05)。相关性分析提示,精子P34H蛋白表达量与HYD活性(HYD阳性反应率、HYD活性强度)存在正相关性,相关系数r=0.449、0.431,P0.01;精子P34H阳性率与HYD活性(HYD阳性反应率、HYD活性强度)存在正相关性,相关系数r=0.727、0.691,P0.01。结论男性不育患者精子P34H蛋白表达减少,精子P34H阳性率降低以及HYD活性(HYD阳性反应率、HYD活性强度)减弱。     

人精子血管内皮生长因子及其受体2的表达和顶体酶活性的关系

目的:探讨人精子血管内皮生长因子(VEGF)及其受体2(VEGFR2)表达和精子顶体酶活性的关系。方法:收集92例精液标本,按照世界卫生组织(WHO)颁布的标准对精液进行常规分析检测。免疫印迹法检测VEGF蛋白在人精子中的表达,免疫荧光法观察VEGFR2在人精子上的定位,采用改良明胶底物膜法检测精子顶体内顶体酶活性(顶体酶阳性反应率、顶体酶活性强度)。结果:免疫印迹检测结果显示,不育各组精子VEGF/GAPDH平均光密度均明显低于正常生育组;免疫荧光染色检测结果显示,不育各组精子VEGFR2阳性率均明显低于正常生育组;不育各组的顶体酶活性(顶体酶阳性反应率、顶体酶活性强度)与正常生育组比较均有显著下降。相关性分析提示精子VEGF蛋白表达量与顶体酶活性存在正相关性;精子VEGFR2阳性率与顶体酶活性存在正相关性。结论:男性不育患者精子VEGF蛋白及其受体2表达降低并伴随顶体酶活性减弱。     

不育男性精子膜蛋白P34H的表达与中性α-糖苷酶活性的关系

目的:探讨男性不育患者精子膜蛋白P34H的表达与精浆中性α-糖苷酶(NAG)活性的关系。方法:收集精液标本,分为正常生育组和不育组。参照WHO标准方法对标本进行精液常规分析,免疫印迹检测P34H蛋白在人精子中的表达,免疫荧光观察P34H蛋白在人精子上的定位,采用底物酶法检测精浆NAG活性。结果:免疫印迹结果显示,正常生育组的精子P34H蛋白表达和精子P34H阳性率均显著高于不育各组;精子P34H蛋白表达及阳性率在NAG活性正常组与NAG活性异常组差异无统计学意义。相关性分析提示精子P34H蛋白表达及P34H阳性率与精浆NAG活性呈正相关性。结论:精子P34H蛋白表达减少、精子P34H阳性率降低以及NAG活性减弱均会导致男性生育力低下,附睾精子蛋白P34H量和NAG活性可用于评价男性生育能力。     

砷对大鼠附睾中肝细胞生长因子及其受体c-met表达与透明质酸酶活性的影响

目的:研究砷对大鼠附睾中肝细胞生长因子(HGF)及其受体c-met与透明质酸酶(HYD)活性的影响。方法:SD大鼠随机分为4组,对照组饮用蒸馏水,低、中、高剂量组分别饮用含2.4、12.0、60.0mg/L亚砷酸钠的蒸馏水。采用免疫印迹法检测HGF、c-met蛋白在大鼠附睾中的表达,改良HYD一明胶底物膜法测精子顶体内HYD活性(HYD阳性反应率、HYD活性强度),扫描电镜观察大鼠精子超微结构的改变。结果:与对照组比较,砷中毒各组HGF、c-met蛋白表达水平均下降,差异有统计学意义;砷染毒中、高剂量组的HYD活性与对照组比较均显著下降。扫描电镜结果显示,与对照组比较,砷染毒低、中、高剂量组大鼠精子顶体完整率均较低,而精子畸形率均较高,差异均有统计学意义。结论:慢性砷中毒能够影响HGF及其受体c-met的表达,使HYD活性减弱,精子顶体完整率下降,精子畸形率升高。     

圆头精子的冷冻保存

目的:探讨圆头精子的冷冻保存效果及其精子功能改变。方法:6例圆头精子症患者各提供1-3份精液标本,应用不含卵黄冷冻保护剂和二步降温方法冷冻保存圆头精子症标本,检测冷冻精子活动率、头-尾膜完整率、存活时间和顶体蛋白酶活性。结果:圆头精子标本冷冻保存后,精子活动率显著减少,膜完整率显著下降,头膜损伤-尾膜完整的精子率显著升高(n=13,P＜0.01)。体外存活时间缩短。精子冷冻前后无顶体蛋白酶活性。不含卵黄冷冻保护剂与含卵黄冷冻保护剂,以及两步冷冻与多步冷冻的效果比较未见显著差异(n=7,P＞0.05)。结论:圆头精子可经历冷冻保存后存活,但冷冻复苏率低。冷冻-解冻过程显著降低精子功能。不含卵黄冷冻保护剂和两步降温可应用于冷冻保存圆头精子症标本。     

冷冻保存对人类精子顶体完整性及超微结构的影响

目的探讨冷冻保存对人类精子顶体完整性及超微结构的影响.方法 2 0例正常生育力精液标本(A组)和27例不育症精液标本(B组)行冷冻保存.应用荧光标记豌豆凝集素法检测冷冻前、后精子顶体完整率.采用透射电镜观察冷冻精子头部超微结构的改变 (n=3).结果解冻后A和B组的顶体完整率与冷冻前的比较均有显著性下降(P<0.01) ,且B组的降低程度明显大于A组.透射电镜观察到冷冻精子头部超微结构发生不同程度的损伤,浆膜和顶体膜出现肿胀、破损,顶体结构异常改变显著增多,顶体内容物丢失,甚至顶体帽缺失.结论冷冻-解冻过程对精子顶体造成了损伤,引起顶体完整率降低和超微结构改变.     

沉默附睾P34H基因对小鼠精子P34H表达和精子透明质酸酶活性的影响

目的:通过慢病毒介导的RNA干扰(RNAi)敲低P34H表达,分析其对小鼠精子P34H表达和精子透明质酸酶(HYD)活性的影响。方法:构建3种附睾精子P34H shRNA慢病毒表达载体GV-P34H-sh RNA-1、GVP34H-sh RNA-2和GV-P34H-sh RNA-3,提取阳性克隆质粒测序,转染HEK293T细胞,生产慢病毒颗粒。将3种重组慢病毒及阴性对照病毒分别注入小鼠附睾中,用real-time PCR和Western blot法分别检测其对P34H m RNA和蛋白的沉默效果。采用免疫荧光法观察P34H蛋白在小鼠精子上的定位,改良透明质酸钠-明胶底物膜法检测小鼠精子HYD活性(HYD阳性反应率和HYD活性强度)。结果:测序结果证实3种慢病毒载体均包装成功。感染小鼠附睾后,P34H的mRNA及蛋白表达量均较阴性感染组和正常对照组明显降低(P0.05);其中以GV-P34H-sh RNA-1作用最为显著,精子P34H阳性表达率和HYD活性均较阴性感染组和正常对照组明显降低(P0.05),而阴性感染组和正常对照组相比差异无统计学显著性。结论:附睾P34H基因沉默可抑制小鼠附睾中精子P34H阳性表达率和HYD活性。     

人精浆中性α-1,4-糖苷酶活性与精子透明质酸酶活性的关系

目的 探讨人精浆中性α-1,4-糖苷酶(中性α-1,4-G)活性与精子透明质酸酶(HYD)活性(HYD阳性反应率及HYD活性强度)的关系.方法 收集年龄24～40岁不育男性精液38例,正常对照12例,根据精浆中性α-1,4-G活性检测结果分为2组:精浆中性α-1,4-G活性正常组(26例)和精浆中性(α-1,4-G活性异常组(12例).24～40岁正常生育男性精液12例作为对照组.用检测盒测定中性α-1,4-G活性,用改良固定底物膜法分别检测精子顶体内HYD阳性反应率及HYD活性强度.结果 不育各组中的精浆中性α-1,4-G活性和精子HYD活性(HYD阳性反应率、HYD活性强度)均显著低于正常生育组(P<0.01).精浆中性α-1,4-G活性与HYD活性(HYD阳性反应率、HYD活性强度)呈显著正相关(r=0.877,r=0.910;P<0.01).结论 精浆中性α-1,4-G活性对精子HYD活性(HYD阳性反应率、HYD活性强度)有明显影响.     

乙酰左旋肉碱对人类精子冷冻前后顶体完整性及超微结构的保护作用探讨

目的 研究乙酰左旋肉碱对人精子冷冻前后顶体完整性及超微结构的保护作用.方法 将18例患者的精液标本液化及PureSperm梯度离心处理后分为2组,分别为A组(对照)和B组(7.5 mmol/L乙酰左旋肉碱),液氮中冷冻2周,比较冻融前后各组精子存活率、 活力、 头部和尾部超微结构损伤比例和顶体完整性.结果 精子冷冻后存活率和活力均较冷冻前明显下降(P<0.05),B组精子解冻后存活率和活力明显高于组A(P<0.05).冷冻后精子头部和尾部损伤比例均较冷冻前明显增加(P<0.05),而B组精子解冻后头部和尾部损伤比例均较A组明显下降(P<0.05).精子解冻后顶体完整性较冷冻前明显下降(P<0.05),B组精子解冻后精子顶体完整性较A组明显提高(P<0.05).结论 冷冻过程对人精子产生损伤,冷冻保护剂中添加7.5 mmol/L乙酰左旋肉碱可以提高精子解冻后顶体完整性,减轻冷冻损伤对于精子头尾超微结构的损伤,起到冷冻保护作用.     

精子形态与顶体酶活性关系的研究

目的探讨精子形态与精子顶体酶活性的关系。方法对395例男性不育患者采用精子形态检测系统下人工修正方法分析精子形态,用酶标法检测精子中的顶体酶和顶体酶活力指数（AAI）。结果精子形态正常组的顶体酶活性明显高于精子形态异常组,两组差异显著（P〈0.05）;顶体酶活性正常组形态正常精子百分率明显高于顶体酶活性异常组形态正常精子百分率,两组间较差异极显著（P〈0.001）;顶体酶活性与形态正常精子百分率间呈显著正相关关系（r=0.169,P〈0.001）。结论精子形态可影响精子顶体酶活性。     

Morphometric analysis of intact sperm heads and of sperm nuclei in the mouse

A morphometric analysis of mouse sperm and of their nuclei was undertaken to investigate their respective post-testicular maturation. Sperm were collected from the testis, caput and cauda epididymidis, and their corresponding nuclei were isolated. Results indicate that the post-testicular maturation of sperm is distinct from that of nuclei. The size of intact sperm heads increases in the caput followed by a subsequent decrease in the cauda. In contrast, sperm nuclei decrease progressively in size. In general, a greater magnitude and number of alterations in intact heads and nuclei occur while in transit from the testis to the caput than during passage to the cauda epididymis. These results suggest that the period immediately following their release from the testis is crucial to the complete morphological maturation of sperm heads and nuclei. © 1993 Wiley-Liss, Inc.     

人精子核完全裂解对RNA提取的影响

目的 证实提取人精子RNA时,精子核完全裂解的重要性.方法 人精子液化后经Pereoll纯化,淋巴细胞分离液分离外周血自细胞用作对照.人精子和外周血白细胞RNA均用TRIzol和RNeasy Kit两种试剂提取,对于不同试剂提取的RNA,取相同细胞数的RNA用于逆转录,并用实时PCR检测两种提取方法的β-ACTIN mRNA 量,来反映所提取RNA的量.结果 RNeasy Kit能完全裂解人精子和外周血白细胞的细胞核,TRIzol能完全裂解外周血白细胞的细胞核,但不能完全裂解人精子核.对于人精子,RNeasy Kit和TRIzol提取RNA量分别为(149.8±24.5)和(35.3±4.0)ng/106精子(n=3),差异有统计学意义(P=0.01).而且这种差异不是由于提取试剂的提取效率造成的,因为对于外周血白细胞,RNeasy Kit提取RNA量还稍低于TRIzol[(765.5±229.8)和(958.8±201.0)ng/106细胞,n=3,P=0.168].结论 人精子核的完全裂解可显著增加精子RNA提取量,也间接说明精子核是人精子RNA存在的重要部位.     

Improvement of sperm motility by the addition of progesterone to the Percoll medium during sperm purification

The Percoll centrifugation technique is widely used to selectthemost motile spermatozoa in connection with assistedre productiontechniques (ART). Progesterone is known toenhance the motilityof spermatozoa in vivo and in vitro. This study evaluates whetherthe motility of spermatozoacan be enhanced by the presence ofprogesterone simultaneously with purification using a routinePercoll centrifugation technique. Motility is measured by computer-assistedspermanalysis. The 100% Percoll medium contained aprogester one concentrationof 2 (ig/ml, which is in the range of that found in pre-ovulatoryfollicular fluid and peritoneal fluid around ovulation. Witha two-layer purification procedure (55/80% Percoll medium),exposure ofthe progesterone in the Percoll medium for only 25mincaused a highly significant improvement of motility in thespermatozoa isolated from the semen samples of 14 healthymen.It is concluded that the addition of progesterone tothe Percollpurification medium can further enhance spermmotility withoutthe need for a change in the routinely used semen purificationtechnique in most assisted reproductionprogrammes.     

Localization of boar sperm proacrosin during spermatogenesis and during sperm maturation in the epididymis

The localization of proacrosin was determined by using colloidal gold labeling and electron microscopy of boar germ cells during spermiogenesis to post-ejaculation. Proacrosin was first localized in round spermatids during the Golgi phase of spermiogenesis; it was associated with the electron-dense granule, or acrosomal granule that was conspicuous within the acrosome. It remained within the acrosomal granule during the cap and acrosome phases of spermiogenesis. At these stages, there was no apparent association of the proacrosin molecule with the acrosomal membranes. During the maturation phase of spermiogenesis, proacrosin was seen to become dispersed into all regions of the acrosome except the equatorial segment. When sperm from different segments of the epididymis and ejaculated sperm were examined, localization was observed throughout the acrosome except for the equatorial segment. Here proacrosin appeared to be localized on both the inner and outer acrosomal membranes as well as with the acrosomal matrix, although further studies are required to verify the membrane localization. No labeling was seen on the plasma membrane. These data suggest that the synthesis and movement of proacrosin to sites in the acrosome are controlled by an as yet unknown process. The absence of proacrosin on the plasma membrane of mature ejaculated sperm makes it unlikely that this enzyme plays a role in sperm-zona adhesion prior to capacitation.     

Effect of abnormal sperm head morphology on the outcome of intracytoplasmic sperm injection in humans

The present study was designed to determine the efficacy of intracytoplasmic sperm injection (ICSI) using spermatozoa with abnormal head morphology in 17 cases with total teratozoospermia. A total of 160 oocytes were retrieved and 144 metaphase II oocytes were injected. The fertilization and cleavage rates were 50.7 and 93.2% respectively. Fertilization failure occurred in two couples. A total of 54 embryos were transferred and pregnancy rates per initiated and per embryo transfer cycle were 17.6 and 20.0% respectively, while the clinical pregnancy rates per initiated and embryo transfer cycle were 11.8 and 13.3%. The implantation rate was 3.7% (2/54). Out of two pregnancies achieved, one resulted in abortion in the first trimester. The ongoing pregnancy rates per initiated and embryo transfer cycle were 5.88% (1/17) and 6.6% (1/15) respectively. Although the implantation and ongoing pregnancy rates are very low, ICSI seems to be the only treatment modality in cases where teratozoospermia was total with 100% abnormal head morphology.      

人类睾丸精子和附睾精子的顶体蛋白酶活性

目的：探讨人类睾丸精子和附睾精子的顶体蛋白酶活性。方法：应用明胶薄层试验、穿卵试验和透射电镜观察顶体蛋白酶活性及其影响。结果：未经洗涤的睾丸精子和附睾精子不出现明显的晕轮，洗涤后则显示出顶体蛋白酶活性。附睾精子组的平均晕轮直径显著高于睾丸精子组（Ｐ＜００１）。附睾精子能与去透明带地鼠卵受精。老化和畸形的附睾精子降低或丢失酶活性。结论：睾丸精子已初步发育具功能活性的顶体蛋白酶，进入附睾顶体蛋白酶逐渐发育至功能成熟，而曲细精管和附睾管内液体存在着抑制酶活性的物质     

Factors influencing human sperm kinematic measurements by the Celltrak computer-assisted sperm analysis system

This study examines the effect of varying several factors, both extrinsic and technology-dependent, on the reconstruction of human sperm trajectories and the derived kinematic measurements using videotapes and the Motion Analysis Celltrak/S instrument. In semen samples from normal healthy men, curvilinear (VCL) and straight line velocities (VSL) were found to increase 1.5-fold, and linearity (LIN) of trajectories and amplitude of lateral head displacement (ALH) increased 1.25-fold when the temperature of analysis was raised from 24 to 37 degrees C. Only VCL and VSL were found to increase significantly between 24 and 37 degrees C for sperm samples selected by Percoll gradient and incubated in a capacitating medium. An analysis chamber of 20 microm depth was found to be adequate for seminal sperm samples while for Percoll-selected sperm samples the analysis in a 50 microm depth provided the highest proportions of spermatozoa with the highest VCL and the largest ALH. The grey level detection threshold required careful adjustment: using a threshold lower than the optimal threshold produced spurious sperm trajectories for seminal sperm samples and rejected some trajectories for Percoll-selected sperm samples. Definition of the appropriate frame rate and maximum burst speed was critical for valid trajectory reconstruction and therefore adequate derived kinematic measurements. Optimal values of these parameters were found to be 30 Hz and 400 microm/s for seminal spermatozoa and 60 Hz and 700 microm/s for selected spermatozoa. The optimal values of 'ALH path-smoothing factor' used to calculate average path and ALH were 5-10 points for seminal spermatozoa analysed at 30 Hz and 15-20 points for selected spermatozoa analysed at 60 Hz. We propose a set of standard conditions for reliable kinematic analysis of human spermatozoa using the Celltrak/S system.      

不同来源精子对ICSI助孕结局的影响

目的探讨不同来源精子对卵胞浆内单精子注射助孕结局的影响。方法回顾分析我中心2008年1月至2012年6月1129个ICSI治疗周期,射出A组858例,附睾穿刺B组229例,睾丸穿刺C组42例,比较3组的临床结局。结果 A组年龄较B、C组大,内膜厚度薄;C组获卵数及ICSI卵数较A、B组多;B组优质胚胎率高于A组,种植率及活产率高于A、C两组（P〈0.05）。通过多元逻辑斯蒂回归模型分析精子来源与活产率的相关性,校正混杂因素后,A组相对于B组的OR值为0.559（95%CI 0.416-0.752）,C组相对于B组的OR值为0.513（95%CI 0.264-0.998）。结论附睾穿刺来源精子行ICSI可获得较好的临床结局。     

The relationship between human sperm apoptosis, morphology and the sperm deformity index

BACKGROUND: This study aimed to assess the relationship between apoptosis in human ejaculated spermatozoa, sperm morphology and the novel sperm deformity index (SDI). METHODS: Semen specimens from 50 healthy donors were prepared by density-gradient centrifugation followed by incubating the prepared sperm with paramagnetic annexin V-conjugated microbeads and subjecting this to magnetic cell sorting (MACS). The procedure delivers two sperm fractions: annexin-negative (non-apoptotic) and annexin-positive (apoptotic). Activated caspase-3 levels and the integrity of the sperm mitochondrial membrane potential (MMP) were assessed as markers of apoptosis in the annexin-negative and -positive aliquots following MACS. Sperm morphology and the SDI scores were assessed using the strict criteria. RESULTS: Compared with the apoptotic sperm subpopulations, the non-apoptotic sperm subpopulations had an improved sperm morphology profile as demonstrated by significantly higher proportions of sperm with normal morphology and significantly lower SDI scores and percentages of sperm with acrosomal defects, midpiece defects, cytoplasmic droplet and tail defects. There was a significant correlation between sperm morphology attributes studied and the expressed apoptotic markers - caspase-3 activation and MMP integrity. CONCLUSIONS: Non-apoptotic sperm fractions have morphologically superior quality sperm compared with apoptotic fractions as reflected by significantly lower SDI scores. The study results may support abortive apoptosis, where the apoptotic mechanism of sperm is already triggered prior to ejaculation.