男性不育精液中尿酸含量与生殖细胞凋亡的探讨

目的探讨人精液中尿酸含量与生殖细胞凋亡的关系.方法参照WHO标准方法,进行精液常规分析,按精子密度、活动率不同分为(正常、＜20、20～40、＞40)4个组.采用尿酸酶-过氧化物酶偶联法检测精液尿酸含量.用脱氧核苷酸末端转移酶(TdT)介导的缺口末端标记(TUNEL)和瑞-姬染色法,分别检测和观察生殖细胞的凋亡.结果75例不育者精液尿酸含量和生殖细胞的凋亡率分别为(263.87±57.15)μmol/L和(16.38±1.25)%与正常生育组(397.60±52.1)μmol/L、(4.61±1.23)%比较呈显著性差异(P＜0.01).精子密度和活动率随精液尿酸含量减少而降低,生殖细胞调亡率随之上升(P＜0.01).不育组精液尿酸含量与生殖细胞的凋亡地显著性负相关(r=-0.93,P＜0.05).凋亡的生殖细胞体积缩小,核染色质致密,凝聚在核周形成新月形,或核裂解形成凋亡小体.结论精液尿酸含量与生殖细胞的凋有着密切关系.低精液尿酸含量可使睾丸生殖细胞凋亡率增加,使精子密度和活率下降而致男性不育.     

人精液一氧化氮含量与男性不育的相关性

目的:研究精液中一氧化氮(NO)含量对精子凋亡的影响及其与男性不育之间的相关性.方法:采用精子质量检测系统对精液标本进行常规检查;应用硝酸还原酶法测定精液中NO的含量;应用瑞-姬染色和TdT介导的原位末端标记(TUNEL)法检测凋亡精子;观察凋亡精子的形态结构改变.结果:不育组精液中NO(58 37±14 14)μmol/l比正常生育组精液中NO(35 20±8 23)μmol/l含量高;正常生育组精子凋亡率9 67%±2 54%比不育组精子凋亡率33 98%±10 54%低.结论:不育组精液中精子的凋亡率随着NO含量的增高而增加.高浓度的NO导致精子凋亡率增加而致使男性生育力下降.     

一氧化氮对大鼠睾丸生殖细胞凋亡作用的研究

目的：探讨一氧化氮（NO）对大鼠睾丸生殖细胞凋亡的影响。方法 采用MTT法观察NO对细胞增殖的抑制作用，透射电镜分析细胞结构变化，末端脱氧核苷酸转移酶（TdT）介导的原位末端标记法（TUNEL）及琼脂糖凝胶电泳检测生殖细胞DNA变化以进一步分析细胞凋亡的特征。结果：MTT法及DNA末端标记法检测表明，NO可抑制细胞增殖，诱导生殖细胞的凋亡，并呈剂量及时间依赖性，NO组细胞凋亡率明显高于对照组（P〈0．01）。透射电镜观察NO处理20h后的生殖细胞，其核染色质凝集、附着于核边缘呈新月形，核固缩、碎裂形成凋亡小体，符合凋亡细胞特征性改变。结论：一氧化氮具有促使生殖细胞凋亡形成的作用，这对不育症的研究有重要的价值。     

男性不育与精子凋亡关系的研究

目的探讨男性不育与精子凋亡的关系。方法采用计算机辅助精液分析对正常生育组（n=20）,不育组（n=60）男性进行精液参数检测,瑞-吉染色检测精子凋亡情况。结果在不同人群中精子凋亡均有一定发生率,正常生育组为（3.52±2.11）%,不育组为（18.26±9.34）%,两者差异极显著（P〈0.01）,并且精子凋亡与精子密度、精子活力以及精子正常形态率呈显著负相关（P〈0.05）。结论精子凋亡与男性不育存在着十分密切的关系,精子凋亡增加,可能是少精症、弱精症和畸形精子症患者不育的原因之一。     

不育男性精液生殖细胞精蛋白的初步研究

在精液常规检查和巴氏染色的基础上按生殖细胞的种类和比例将６４例不育患者分为３型。分别提取生育与不育男性精液生殖细胞核内碱性蛋白，经氨基酸分析表明正常生育对照组合有约４７．４％的精蛋白成份。高效液相色谱分子筛分析生育与不育男性精液生殖细胞核碱性蛋白发现：１４例生育男性均存在精蛋白，其含量约占核碱性蛋白的３６．７８％～６４．４２％；５６例有精子和其它生精细胞的Ⅰ型和Ⅲ型不育患者中，精蛋白缺乏者５例，占８．９％。精蛋白含量减少者１１例，占１９．６％。精蛋白含量在正常生育男性范围内波动者４０例，占７１．４％；８例无精子的Ⅱ型不育患者均缺乏精蛋白。     

睾丸生殖细胞凋亡及其调控因素研究进展

1972年Kerr^[1]等发现细胞的自发性死亡分为坏死和凋亡两种类型。细胞凋亡是组织对各种刺激的主动反应过程，不引起炎症反应。在此过程中，由于内源性核酸内切酶活性增强，在核小体之间特异地剪切DNA，形成大小约180～200bp倍数的DNA片段，从而形成了细胞凋亡的特征性改变。正是由于这种特征性改     

NO与心血管疾病细胞凋亡

NO是重要生物活性物质,介导着心肌、血管平滑肌、巨噬细胞、血管内皮细胞等程序性死亡与凋亡,与心血管疾病有密切的关系.并对NO与心血管疾病细胞凋亡的机制和目前研究方向及存在问题进行了文献综述.     

NO与细胞凋亡

NO对不同细胞的凋亡影响不同.NO诱导凋亡可通过氧化应激、干扰能量代谢、直接损害DNA、激活多聚ADP-核糖聚合酶、或使胞液Ca2+调节紊乱.而NO抑制细胞凋亡通过cGMP依赖途径、细胞色素C释放抑制、半胱天冬酶酶活性降低等来实现的.     

饮酒与男性不育的关系研究

目的探讨长期高浓度过量饮酒者精液中一氧化氮（NO）含量其精子质量和生精细胞凋亡与不育的关系。方法随机将146例精液分为5组,采用硝酸还原酶法特异地将NO代谢产物硝酸盐（NO3^-）测还原成亚硝酸盐（NO2^-）总量代表NO水平。用脱氧核苷酸末端转移酶（TdT）介导的缺口末端标记（TUNEL）法和双目光学显微镜,分别检测和观察生精细胞的凋亡率及形态结构。用SQA-V全自动精子质量分析仪,测定精子质量。结果未饮酒生育组精液中NO的含量为（54.81±11.45）μmol/L,生精细胞的凋亡率（4.52±1.23）%,精子质量活率（80.24±0.17）%、活力a＋b（78.32±0.12）%、畸形率（5.30±0.13）%,与长期高浓度过量饮酒各组结果相比,均呈显著性差异（P〈0.01,见表2）。长期高浓度过量饮酒各组NO的含量和生精细胞的凋亡率呈显著的正相关（r=0.93）。凋亡的生精细胞核染色质浓缩于核周形成新月形,核裂解形成凋亡小体。结论长期高浓度过量饮酒者精液中NO的含量和生精细胞的凋亡率均升高,精子质量低下。提示长期高浓度过量饮酒可致机体NO过度产生而促使生精细胞凋亡致男性不育的因素之一。     

男性不育患者9号染色体异常与精液常规结果关系的探讨

目的探讨男性不育患者9号染色体异常和精液常规结果之间的关系。方法对3638例男性不育症患者的精液常规检查和外周血淋巴细胞染色体结果进行回顾性分析。结果 52例男性9号染色体异常,占不育症患者的1.43%,精子密度、精子活率、A级精子、B级精子与正常对照组没有明显的区别（P〉0.05）。结论 9号染色体异常与男性精液检查结果异常没有直接的关系。     

Carcinoma in situ of the testis: frequency and relationship to invasive germ cell tumours in infertile men

A light microscopical study on a total of 812 consecutive testicular biopsies from 555 infertile men revealed intratubular changes in germ cells compatible with a carcinoma in situ pattern in six oligospermic patients (I.I%); the changes were found in both testes in two of these men. Four of the six patients developed an invasive germ cell tumour within follow-up period of 1.3 to 4.5 years. The results confirm the malignant nature of these intratubular atypical germ cells. It is concluded that testicular biopsy may be useful for early detection and cure of germ cell carcinoma in patients at risk, i.e. patients with cryptorchidism, infertile men or patients with previous cancer of one testis.     

Frequency of hyper-, hypohaploidy and diploidy in ejaculate, epididymal and testicular germ cells of infertile patients

The hypothesis that sperm aneuploidy and diploidy increase as a function of spermatogenesis impairment was addressed. Ejaculated semen samples from a series of men (n = 22) with very low total normal motile count (1 x 10(6)) was analysed in terms of sperm aneuploidy and diploidy by in-situ hybridization and compared with controls (n = 10). Germ cell aneuploidy was also analysed in an additional series of infertile patients presenting unexplained infertility (n = 3), congenital absence of the vas deferens (CAVD) (n = 6) and non-obstructive azoospermia (n = 3) undergoing IVF, microsurgical epididymal sperm aspiration (MESA)/ICSI and testicular sperm extraction (TESE)/ICSI cycles respectively. In-situ hybridization for chromosomes 1, 17, X and Y was performed on ejaculate, epididymal and testicular spermatozoa. Significantly higher sperm aneuploidy and diploidy rates where found (for the four chromosomes analysed) in spermatozoa from oligoasthenoteratozoospermia (OAT) over controls (18 versus 2.28% and 2.8 versus 0.13% respectively; P < 0.001). Testicular germ cells had even higher rates of sperm aneuploidy and diploidy. However, in this group it was difficult to determine whether the cells analysed were dysmorphic spermatozoa or spermatids. The data warrant further investigation on the cytogenetic abnormalities found in most germ cells identified in testicular tissue biopsies of azoospermic patients.     

缺氧诱导的心肌细胞凋亡及一氧化氮的保护作用

目的　研究缺氧在新生大鼠心肌细胞凋亡中的作用及一氧化氮 (NO)的保护作用。方法　取体外培养的新生大鼠心肌细胞置 95 0mL/LN2 ,5 0mL/LCO2 孵箱中培养16 ,32和 48h ,造成缺氧损伤的细胞模型 ,观察凋亡发生的情况 ;将NO供体SNAP加入培养基中 ,使其终浓度为 10 0μmol/L ,置于上述缺氧环境中培养 16 ,32和 48h ,检测细胞凋亡。结果在缺氧条件下培养 16 ,32 ,48h后 ,心肌细胞的凋亡率分别为 :( 2 9± 0 5 ) % ,( 6 2± 0 8) %和 ( 2 6 6±3 0 ) % ;而加入NO供体后 ,凋亡率分别为 :( 0 2± 0 3) % ,( 3 4± 0 4) %和 ( 11 8± 1 2 ) %。结论凋亡是心肌细胞缺氧损伤的主要形式 ;NO对缺氧引起的心肌细胞凋亡有保护作用     

Correlation between semen parameters and sperm aneuploidy rates investigated by fluorescence in-situ hybridization in infertile men

Spermatozoa from 32 infertile patients and 13 controls with normal semen parameters were analysed using dual and triple colour fluorescence in-situ hybridization (FISH) techniques, in order to investigate the rates of aneuploidy for chromosomes 13, 18, 21, X and Y. The patients were divided into three groups according to their karyotypes or the karyotypes of their offspring: 15 were infertile men with abnormal semen parameters and normal karyotypes (group 1), 13 were infertile men with abnormal karyotypes and normal or abnormal semen (group 2) and four were infertile men with abnormal semen and normal karyotypes but whose wives conceived a child (or a fetus) with a numerical chromosomal abnormality through an intracytoplasmic sperm injection cycle (group 3). Patients with abnormal semen parameters showed a significantly higher aneuploidy rate for the investigated chromosomes in their spermatozoa compared to controls (P < 0.005). Our data suggest the presence of a correlation between poor semen parameters and an increase in aneuploidy rate of chromosomes 13, 18, 21, X and Y in spermatozoa (r = -0.81071, P < 0.002); therefore the risk of a chromosomal aneuploidy in spermatozoa seems to be inversely correlated to sperm concentration and total progressive motility. Patients with abnormal karyotypes showed a higher incidence of diploidy and chromosomal aneuploidies compared to controls (P < 0.002). This strongly suggests the presence of an interchromosomal effect of the cytogenetic rearrangement. Men who fathered a child with an abnormal karyotype through intracytoplasmic sperm injection did not present a higher aneuploidy rate for the investigated chromosomes in spermatozoa compared to patients with infertility due to a similar male factor but showed higher incidence of chromosomal aneuploidy compared to normal controls.     

NO介导的小鼠胸腺细胞中线粒体的变化在细胞凋亡过程中的作用

目的研究一氧化氮(NO)介导的胸腺细胞凋亡中线粒体膜电位(ΔΨm)和心磷脂(CL)含量变化的特点。方法以S-亚硝基-N-乙酰青霉胺(SNAP)作为NO的供体诱导胸腺细胞凋亡,以地塞米松(DEX)作为阳性对照药物;设空白对照组、SNAP组和DEX组3个实验组;经膜联蛋白V(annexinVmAb)和碘化丙啶(PI)染色后,用流式细胞术(FCM)检测细胞磷脂酰丝氨酸(PS)外翻;用3,3’-二已基噁羰花青碘化物[DiOC6(3)]和PE-anti-annexinVmAb检测凋亡中ΔΨm变化;用壬基吖啶橙(NAO)和PE-anti-annexinVmAb检测凋亡中线粒体CL变化。结果SNAP作用后6h,胸腺细胞出现典型的细胞凋亡特征,多数annexinV阳性的细胞出现皱缩。DEX组ΔΨm降低且未凋亡的细胞比例显著高于空白对照组(P<0.01);而SNAP组该群细胞所占比例与空白对照组比较,差异无统计学意义(P>0.05)。各组中约40%~50%的DiOC6(3)阴性细胞同正常细胞的大小。SNAP组CL含量降低的凋亡细胞所占比例显著高于对照组(P<0.01),未见CL含量降低且未凋亡的细胞群。空白对照组和SNAP组中分别有(48.32±3.96)%、(43.64±4.90)%的细胞CL含量降低但大小同正常细胞。结论NO介导的小鼠胸腺细胞的凋亡过程,依次为磷脂酰丝氨酸外翻、线粒体去极化、CL氧化及细胞皱缩。同DEX模型组相比较,NO介导的小鼠胸腺细胞线粒体的变化为凋亡过程中较晚期的变化。     

Human seminal plasma inhibits brain nitric oxide synthase activity

Nitric oxide is a chemical messenger which functions as a neurotransmitteror as a cytotoxic agent. Nitric oxide synthase (NOS) has beenisolated from various mammalian reproductive tissues* The presenceor absence of NOS in spermatozoa has not yet been reported.We therefore tested human and marine spermatozoa for NOS activityby measuring the conversion of argmine to citmlline. No activitywas found either in human or in murine spermatozoa. Human nativesemen and human seminal plasma exerted an inhibition on brainNOS activity, as assayed on rat brain cytosolic fractions. Thisinhibitory effect was dependent on the amount of protein presentin the human seminal plasma. No inhibitory effect was observedwhen homogenates of washed spermatozoa were tested. The humanseminal plasma did not affect the Michaelis constant (Km) ofNOS for L-arginine (endogenous NOS substrate) whereas the maximalvelocity (Vmax) was reduced, suggesting that it contains a non-competitiveinhibitor of brain NOS. This inhibitory component was virtuallyinsensitive to heat; a 10 min treatment to 95°C only slightlyreduced its ability to inhibit brain NOS. The physiologicalrelevance of our observations remains to be elucidated. Humanseminal plasma may exert an inhibition of nitric oxide synthesison cells other than spermatozoa or on cells from the male orfemale genital tract, modulating directly or indirectly (viamodulation of reactive oxygen species formation) the functionalstate of the spermatozoa.     

一氧化氮与白细胞精子症不育的关系探讨

目的　探讨一氧化氮 (NO)与白细胞精子症不育的关系。方法　参照WHO标准方法 ,进行精液常规分析。采用过氧化物酶染色法检测精液中白细胞密度。用改良的低渗肿胀法检测精子细胞膜的完整性。采用镀铜镉还原荧光法 ,检测精液中NO代谢产物硝酸盐 (NO 3 )。结果　白细胞精子症不育组白细胞的密度为 (1.4 8± 0 .90 )× 10 9/L ,NO水平为 (10 3.5± 2 .0 ) μmol/L ,显著高于正常生育组的(0 .73± 0 .2 8)× 10 9/L和 (41.6± 1.8) μmol/L (P分别为<0 .0 5和 <0 .0 0 1)。精子的活动率、精子速度试验 (SVT)及精子头、尾部膜完整率 ,均明显低于生育组 (P <0 .0 1) ,而精子的死亡率则显著高于生育组 (P <0 .0 1)。结论　NO水平与白细胞密度呈正相关 ;与精子的活动率、SVT及精子头、尾膜的完整率呈负相关。表明当精液中的白细胞增高时 ,可产生过量的NO导致精子中毒受损使精子的受精能力下降     

精子运动能力与一氧化氮关系的探讨

目的探讨一氧化氮(NO)与精子运动能力的关系.方法采用镀铜镉还原荧光法,测定人精液中NO代谢产物硝酸盐(NO3-).参照WHO方法,在超高倍显微镜下观察精子存活率、活动力等. 结果 80例男性不育者其中15例NO浓度明显低于正常对照组精子活动力a b级<50%(P<0.01),以运动轨迹异常和头摆动幅度下降为主, 精子存活力>75%无明显差异(P>0.05);65例NO浓度显著高于正常对照精子活动力a b 级<50%,存活率<75% (P<0.001).结论 NO与精子运动功能有着密切关系.高浓度时明显抑制精子活动力及存活率.低浓度时精子存活率及功能有着维护作用,这对男性不育的病因研究和治疗有非常重要的临床价值.     

Localization of nitric oxide synthase in human trophoblast cells: role of nitric oxide in trophoblast proliferation and differentiation

PROBLEM: There are conflicting reports about the isoform of nitric oxide synthase (NOS) present in trophoblast cells. In this study, we have examined the presence of different NOS isoforms in trophoblast cells. In addition, the role of nitric oxide (NO) in trophoblast function has also been studied by investigating the possible role of nitric oxide in trophoblast proliferation and differentiation. METHOD OF STUDY: NOS isoforms in primary-term trophoblast and JEG-3 cells were identified by immunocytochemistry. The intracellular localization of this enzyme was determined by confocal laser scanning microscopy. Trophoblast proliferation was studied by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrasolium bromide (MTT) conversion assay and cellular differentiation was monitored by human chorionic gonodotropin (hCG) and progesterone secretion, measured by radioimmunoassay. RESULTS: The immunoreactive NOS was present in human trophoblast cells of normal term placenta and JEG-3 cells (a choriocarcinoma cell line) maintained in culture. Nicotinamide adenine dinucleotide phosphate (NADPH)-dependent diaphorase activity overlapped with the immunostaining of NOS. Specific antibodies against the different isoforms of NOS detected the presence of neuronal-type NOS (nNOS) only. The other two isoforms, i.e., eNOS (endothelial) and iNOS (macrophage specific) were completely absent. The nNOS was localized in cell cytoplasm. In culture, JEG-3 cells normally undergo proliferation and cytotrophoblast cells in primary culture differentiate to form hormone-secreting syncytial cells. Sodium nitroprusside (SNP), a nitric oxide donor, when added to the culture, significantly increased proliferation of JEG-3 cells and inhibited the differentiation of cytotrophoblast cells. The arrest by SNP in the formation of syncytial cells was further evidenced by the low secretion profile of hCG and progesterone. CONCLUSIONS: Our findings suggest for the first time the presence of nNOS in the human trophoblast cells and a previously unrecognized role of NO in trophoblast proliferation and differentiation.