男性不育患者精浆NO水平及其与抗精子抗体的关系

目的 :观察男性不育患者精浆中 NO水平变化及其与 As Ab的关系。方法 :采用EL ISA法测定了 1 1 6例男性不育患者及 49例正常生育男性血清和精浆 As Ab,同时采用硝酸还原酶法测定 1 1 6例男性不育患者与 49例正常生育男性精浆中 NO水平。结果 :男性不育患者 As Ab阳性率 3 8.8% ,显著高于正常生育组 4.0 8% ( P<0 .0 1 )。不育组精浆中 NO水平 1 4 2 .8± 3 9.2 μmol/ L,显著高于正常生育组 83 .9± 2 6 .1 μmol/ L( P<0 .0 1 )。As Ab阳性的不育患者 NO水平 1 59.3± 42 .6μmol/ L ,显著高于 As Ab阴性组 1 3 4 .2±3 1 .4μmol/ L ( P<0 .0 1 )。结论 :As Ab与男性不育有着密切相关性 ,且为重要的免疫因素 ,男性不育患者精浆内高水平的 NO与 As Ab的产生可能有着密切的关系。     

整合素亚基α_5、β_1在人精子的表达及其与受精的关系

目的 :研究精子表面整合素亚基 α5、β1的表达及其与精子功能状态和受精力的关系 ;整合素亚基 α5、β1的表达与不明原因不育症的关系。方法 :1 3例生育力正常男性的精液标本 ,9例临床诊断为不明原因不育症患者的精液标本。以精子穿透去透明带的金黄地鼠卵行体外受精试验 (SPA)检测精子受精力 ;对新鲜、获能和孕酮诱导顶体反应后精子行间接免疫荧光染色 ,流式细胞仪检测精子表面整合素亚基α5、β1表达阳性的精子百分率。以三色法染色 ,观察精子顶体反应的发生率。结果 :流式细胞仪检测显示正常组新鲜精子表面α5亚基的表达 (1 3 .3± 5 .4% )与对照组 (1 0 .7± 6 .4% )无显著差异 (P>0 .0 5 )。获能组精子表面 α5亚基的表达率 (6 5 .7± 1 7.6 % )比新鲜组和对照组显著增高 (P<0 .0 5 )。孕酮诱导顶体反应后 ,α5亚基的表达率 (6 5 .3± 1 8.9% )比新鲜组和对照组显著增高 (P<0 .0 5 ) ,但与获能精子组相比无显著差异 (P>0 .0 5 )。β1亚基的表达率分别为新鲜组1 2 .9± 7.4% ,获能组 1 5 .9± 7.9% ,诱导顶体反应组 1 6 .9± 6 .2 % ,与对照组 (分别为 :9.3± 3 .4% ,1 7.3± 7.8% ,1 2 .2± 8.7% )比较均无显著差异 (P>0 .0 5 )。获能精子表面α5亚基的阳性表达率与受精率有一定的线性相关 (r=0 .     

血管紧张素Ⅱ对人精子顶体反应的影响

目的 :探讨血管紧张素 (angiotensin ,Ang )对人精子顶体反应的影响及其可能机制。方法 :检测不同浓度 Ang 诱发的人精子顶体反应率和 Ang 引起的精子胞内钙离子的变化 ,以及血管紧张素受体 AT1拮抗剂 Losartan对其的抑制作用。结果 :Ang 在 1 0 nmol/L和 1 0 0 nmol/L时均可显著增加人精子顶体反应率 ,并引起精子胞内钙离子短暂快速升高 ,Losartan可以明显抑制这些过程。结论 :Ang 可以诱发人精子顶体反应 ,这一作用可能经 AT1受体介导 ,通过引起精子胞内钙离子升高而实现。     

不同人群体内抗精子抗体的初步观察

目的 :进一步探索 As Ab与生殖的关系。方法 :本文应用 EL ISA间接法对 1 4 0对不育夫妇的血清、2 9例初孕妇、6 5例未婚青年、2 57例 2 5岁～ 72岁已育者的血清作了抗精子抗体( As Ab)的检测 ,并用同法对 1 4 0例不育丈夫的精浆也作了 As Ab检测。结果 :不育夫妇血清As Ab阳性率男方为 3 0 %、女方为 2 2 .8%、精浆 As Ab阳性率为 1 3 .6 %。其血清 As Ab与未婚青年和初孕妇相比均有显著差异 ,与同龄生育者相比男性有显著差异 ,女性无显著差异。生育者血清 As Ab阳性率男性随年龄增长呈上升趋势 ,女性则无明显差异。结论 :人体内 As Ab与不育有一定关系 ,老年男性 As Ab可能作为一种自身抗体与机体免疫调节功能有关。     

囊性纤维化跨膜转导调节因子(CFTR)表达率与人精子获能及顶体反应间相关性研究

目的:研究人精子囊性纤维化跨膜转导调节因子(cystic fibrosis transmembrane conductance regulator,CFTR)表达率与精子获能及顶体反应间的相关性。方法:通过间接免疫荧光染色法观察CFTR在人精子上的表达率,金霉素染色法评估精子获能及顶体反应。结果:CFTR定位在人精子赤道板上,随着CFTR表达率降低,精子获能率也随之降低,故CFTR表达率与人精子获能呈正相关(r=0.985,P0.01),而且精子顶体反应率也随之减少,因此CFTR表达率与人精子顶体反应呈正相关(r=0.979,P0.05)。结论:人精子CFTR表达率与精子获能及顶体反应呈正相关。     

一氧化氮与大鼠离体黄体细胞凋亡的关系及白细胞介素-1β对其影响

目的 :探讨一氧化氮在黄体退化中的作用及细胞因子对其影响。方法 :以孕马血清促性腺激素 -人绒毛膜促性腺激素处理的雌性 Wistar大鼠为模型 ,分离制备了黄体细胞。采用 3'-末端标记 (TUNEL)法分析了 NO供体硝普钠 (SNP)、白细胞介素 -1 β(IL-1 β)对离体黄体细胞凋亡的作用。应用比色法及高效液相色谱仪分别检测了 IL-1 β对大鼠离体黄体细胞 NO合酶 (NOS)活性和 NO释放量的影响。结果 :SNP可以剂量方式诱导黄体细胞凋亡 ,1 μmol/ L SNP即可显著增加大鼠离体孵育 1 2 h的黄体细胞凋亡率 (为对照组的 1 33±1 8% ,P<0 .0 5)。IL-1 β(1 0 ng/ ml)可增强 SNP的作用。此外 ,IL-1 β可显著增加离体孵育1 2 h黄体细胞的总 NOS活性 (由 0 .2 0 1± 0 .0 51 U/ mg protein增高到 0 .382± 0 .1 0 6 U/mg protein,P<0 .0 1 ) ,促进 NO生成。结论 :NO-NOS系统可能通过诱导黄体细胞凋亡的途径参与黄体退化过程 ,IL-1 β可增强 NO-NOS系统的活性     

Na~+/K~+-ATP酶——影响精子运动的重要因素

Na+/K+-ATP酶是存在于几乎所有真核生物细胞膜上的一种跨膜蛋白质,是维持细胞内环境离子稳定及细胞生命活动重要的蛋白酶。其主要的催化亚基α亚基有4个亚型:α1、α2、α3和α4,其中α4亚基是精子特异性亚基,可能直接参与精子运动能力的调节,对精子运动起至关重要的作用。而Na+/K+-ATP酶的天然抑制剂Ouabain能够通过Na+/K+-ATP酶介导的多种途径如NHE(Na+/H+-Exchanger)学说、血流动力学说、膜电位学说、信号转导学说等使精子运动能力降低。临床上弱精子症、严重少精子症、阳痿、早泄等性功能障碍性疾病以及不射精症、逆行射精等射精障碍性疾病导致的男性不育也可能与Na+/K+-ATP酶的异常有关。     

血管紧张素Ⅱ对人精子获能的影响

目的:探讨血管紧张素II(angiotensinII,AngII)对不育症精子获能的影响及其可能的作用方式。方法:不同浓度AngII与精子悬液共同孵育,以孕酮诱发顶体反应,采用FITC-PSA法测定细胞内Ca2+荧光强度来检测。结果:在未获能的精子悬液中AngII在10nmol/L和100nmol/L时均可明显加速精子获能和增强孕酮诱发的顶体反应(P<0.05),并可使细胞内Ca2+浓度升高(P<0.001),Losartan[AngII受体AT1(angiotensinIItype1receptor)拮抗剂]、EGTA可明显阻断和抑制这些过程。结论:AngII可以促进不育症精子获能,其结果可能是通过AT1受体介导,经改变精子细胞内Ca2+的浓度来实现。     

微电脑脉冲式水囊按摩药渗仪治疗慢性细菌性前列腺炎性不育症的探讨

目的 :探讨自制的微电脑脉冲式水囊按摩药渗仪 ( MPWMDP仪 )治疗慢性细菌性前列腺炎性不育症 ( ICCBP)的临床效果。方法 :96例 ICCBP病人给予尿道内药物灌注、微电脑前列腺小管药物渗透及前列腺脉冲式水囊按摩治疗。 64例 ICCBP病人给予口服药物为对照。结果 :治疗组治愈并使配偶受孕 66例 ( 68.8% ) ,总有效 92例 ( 95 .8% ) ,分别高于对照组的 1 1例 ( 1 7.2 % )和 5 1例 ( 79.7% ) ( P<0 .0 1 )。治疗组精子密度、活率、精浆锌 ( Zn)和超氧化物歧化酶 ( SOD)含量及 As Ab阴转率均明显优于对照组 ( P<0 .0 1 )。前列腺液白细胞 ( EPS- WBC)与精子密度、活率、精浆 Zn和 SOD含量之间均呈显著负相关 ( P<0 .0 1 )。结论 :MPWMDP仪对 ICCBP是有效、安全、科学的治疗新途径     

固相透明质酸法优选精子的效果观察

目的：对比研究高通量的固相透明质酸（HA）分离新技术较传统密度梯度法在精子优化分离效果方面的优势性。方法：收集新鲜精液标本31份,每份标本液化后被均分成2部分,一部分精液以固相HA法分离精子,即将精液加入到底部表面包被有HA的培养皿中,洗脱掉未结合的精子,分离、收集与HA结合的精子：另外一部分精液以传统的密度梯度法优化分离精子,观察2种方法提取后精子各项研究指标的差异性,以及相对变化幅度。结果：与密度梯度法组相比,固相HA法组处理后精子的正常形态率、获能2h酪氨酸磷酸4~（TP）、ATP（精子获能2hTP率一未获能精子TP率）、诱发顶体反应（AR）和△AR（诱发AR率一自发AR率）等指标明显增高CP〈0．001）,增长幅度依次为：1．55倍（95％CI=1．04,~2．81）、1．81-fg-~（95％CI=0．89-6．11）、3．35-9}（95％CI=1．04-10．32）、1．37倍（95％CI=0．96～2．71）和1．88倍（95％CI=1．09,-4．71）;而固相HA法组自发AR、核蛋白不成熟度和DNA碎片率显著降低（P〈O．001）,分别是密度梯度法组的68％（95％CI=19％-102％）、47％（95％CI=2~／~103％）和21％（95％CI=0％～70％）;2种方法处理后精子的前向运动率和活动率无统计学差异（P〉0．05）。结论：基于成熟精子头部具有HA受体可与外源性HA特异性结合原理的精子。固相HA分离技术,与传统的密度梯度法相比,分离后的精子具有更优的受精潜能和成熟度,可显著提高分离精子的整体质量,有望进一步改善辅助生殖结局。     

Effect of sperm-immobilizing antibodies on the acrosome reaction of human spermatozoa.

OBJECTIVE: To study by a triple stain technique the effect of sperm-immobilizing antibodies on the acrosome reaction of human spermatozoa. DESIGN: The spermatozoa were allowed to swim up and culture in a medium containing 7.5% (vol/vol) serum with sperm-immobilizing antibodies or control serum up to 6 hours. Sperm mobility was analyzed, and the percentage of live acrosome reacted spermatozoa was determined. SETTING: Samples were collected from patients referred to university hospital infertility clinics. MATERIALS: Serum samples were drawn from seven patients with sperm-immobilizing antibodies. All the sera were heat activated and stored at -40 degrees C until use. Semen samples were taken from two healthy donors. RESULTS: During culture for 6 hours, the percentage of live sperm showing the acrosome reaction increased significantly (P less than 0.01) in the control group but not in the sperm-immobilizing antibodies group. However, the inhibitory effect of sperm-immobilizing antibodies on the acrosome reaction was reversed when sperm was reincubated in medium with control serum (P less than 0.01). CONCLUSIONS: Sperm-immobilizing antibodies block fertilization at least in part by inhibiting the acrosome reaction of human spermatozoa.     

The kinetics of the acrosome reaction of human spermatozoa and its correlation with in vitro fertilization.

OBJECTIVE: To study the reaction pattern of acrosome reaction in human semen and correlate it to the results of in vitro fertilization (IVF). DESIGN: The percentage of acrosome-reacted spermatozoa of 41 IVF semen samples was determined after 0, 2, 4, and 24 hours of incubation in human tubal fluid medium supplemented with 10% human pool serum. SETTING: St. Radboud Hospital, Catholic University of Nijmegen, The Netherlands. PATIENTS: Forty-one IVF couples. INTERVENTIONS: None. MAIN OUTCOME MEASURE: Acrosome reaction was determined using fluorescein isothiocyanate conjugated concanavalin A lectin. To avoid false-positive signals from dead spermatozoa, the sperm viability was determined. RESULTS: Three kinetic patterns of acrosome reaction could be distinguished: (1) normal reacting pattern (percentage of acrosome-reacted spermatozoa less than 10% at 2 hours and greater than 5% at 4 hours; 75% fertilization in IVF); (2) a quickly reacting pattern (percentage of acrosome-reacted spermatozoa greater than 10% at 2 hours; 22% fertilization in IVF); and (3) a nonreacting pattern (percentage of acrosome-reacted spermatozoa less than 5% at all time intervals studied; 15% fertilization in IVF). CONCLUSIONS: The timing of acrosome reaction and the percentage of acrosome-reacted spermatozoa are very important parameters in IVF.     

Effect of swim-up sperm washing and subsequent capacitation on acrosome status and functional membrane integrity of normal sperm

OBJECTIVE: Sperm preparation techniques select sperm population with improved sperm motion characteristics. We sought to determine whether the swim-up technique selects spermatozoa with the ability to undergo hypoosmotic swelling, and how swim-up and subsequent capacitation affect the acrosome reaction rate. METHODS: Semen specimens from 15 normal donors were divided into unprocessed, swim-up, and capacitated groups, and sperm motion characteristics, ability to undergo hypoosmotic swelling, and acrosome reaction rate were measured. RESULTS: Sperm motility, viability, and all motion characteristics (except linearity) were significantly increased in both swim-up and capacitated specimens. The ability to respond to hypoosmotic swelling was significantly higher in the spermatozoa isolated by swim-up. The percentage of acrosome-reacted spermatozoa remained unchanged in both unprocessed and swim-up groups, but was significantly higher in the capacitated group. CONCLUSIONS: Swim-up isolates sperm with greater ability to undergo hypoosmotic swelling, but does not change the acrosome reaction rate. In vitro capacitation of spermatozoa selected by swim-up enhances the acrosome reaction rate.     

High progesterone concentrations induce acrosome reaction with a low cytotoxic effect.

OBJECTIVE: To determine the optimal conditions to obtain live acrosome-reacted spermatozoa for micromanipulation. DESIGN: Experiments were performed to determine time and dose-dependent effects of calcium ionophore A23187 or steroids on acrosome reaction of fertile donor sperm. The percentages of total reacted and live reacted spermatozoa were assessed with the peanut agglutinin lectin procedure. RESULTS: Incubation with 1 mmol/L progesterone (P) induced 48% +/- 17% acrosome reaction after 6 hours. Motility and viability remained high (49% +/- 3% and 70% +/- 2%, respectively) and thus the percentage of live reacted spermatozoa was 27% +/- 5%. Incubation with A23187 (5 mumol/L for 30 minutes) gave similar results for the percentage of live reacted spermatozoa (26% +/- 4%) but with a lower motility and viability (25% +/- 7% and 53% +/- 2%, respectively; P less than 0.05). CONCLUSIONS: These results show that high concentration of P is an effective way to induce acrosome reaction in preparation for micromanipulation.     

γ－氨基丁酸诱发人精子顶体反应及其对若干离子转运影响的研究

本文用Ｌｅｃｔｉｎ染色法、原子吸收光谱法和电化学法研究了γ－氨基丁酸对人类精子顶体反应的诱导作用，以及它对Ｎａ＋、Ｋ＋、Ｃａ２＋、Ｍｇ２＋和Ｃｌ－在顶体反应时的转运情况的影响。结果表明：１．γ－氨基丁酸可明显地诱发人精子顶体反应（Ｐ＜０．０５）。２．γ－氨基丁酸可引起Ｎａ＋、Ｃａ２＋、Ｍｇ２＋内流，Ｋ＋外流和Ｃｌ－先外流再内流。因此，γ－氨基丁酸对人精子具有调控作用。     

Defective function of a nongenomic progesterone receptor as a sole sperm anomaly in infertile patients.

OBJECTIVE: To compare the function of a novel nongenomic progesterone (P) receptor on the human sperm surface (mediating the P-induced acrosome reaction) in spermatozoa from fertile donors and from infertile patients. To examine the possible implication of defective P receptor function as an etiologic factor in unexplained male infertility. DESIGN: Progesterone binding and P effects were assessed in sperm from infertile patients and compared with corresponding parameters for sperm from healthy donors. SETTING: Private hospital, medical research center, and a university-based andrological laboratory. PATIENTS, PARTICIPANTS: Sperm samples were from infertile patients (no pathology detected in their wives) attending our infertility clinic and from healthy sperm donors. INTERVENTIONS: None. MAIN OUTCOME MEASURES: Progesterone binding sites were visualized with a fluorescein-labeled protein-P conjugate. Indo 1-AM (a fluorescent indicator of intracellular free Ca2+) was used to measure P-induced Ca2+ influx. Progesterone-induced acrosome reaction was monitored after acrosomal staining with Pisum sativum agglutinin. RESULTS: Among 8 patient sperm samples with normal spermiogram values (of 53 examined), 5 showed a reduced percentage of P-binding spermatozoa and an abnormal response to the hormone in terms of Ca2+ influx and the acrosome reaction. CONCLUSIONS: Defective function of a sperm surface P receptor is described in some cases of male infertility. The observed fluorescence microscopic patterns of hormone binding may be used to further investigate receptor activity in unexplained male infertility.     

Direct effects of progesterone and antiprogesterone on human sperm hyperactivated motility and acrosome reaction.

OBJECTIVE: To study the direct effects of progesterone (P) and its antagonist RU486 (mifepristone) on sperm hyperactivation (HA) and acrosome reaction. DESIGN: Prospective evaluation of semen samples incubated in capacitation media with P and/or RU486. SETTING: University-affiliated tertiary care center. PATIENTS: Normal healthy volunteers. INTERVENTIONS: Semen samples were incubated in media with P or RU486 alone or in combination, and aliquots were taken at 10 minutes, 1, 5, and 24 hours for HA analyses by computer-aided sperm analysis system, and at 0, 5, and 24 hours for assessment of acrosome reaction by fluorescein-labeled Pisum sativum (pea) agglutinin. MAIN OUTCOME MEASURES: HA and acrosome reaction. RESULTS: Sperm HA was significantly increased at 10 minutes by P both at 10(-7) M (9.27 +/- 1.59%; mean +/- SEM) and 10(-6) M (9.39 +/- 1.94%) when compared with untreated spermatozoa (5.62 +/- 1.59%). The stimulatory effect of P on sperm HA was transient because this was not observed after 1, 5, and 24 hours of incubation. The antiprogesterone RU486 (10(-6) M) alone had no effect and did not abolish the stimulatory effect of P on HA. The %HA was further enhanced by the addition of RU486 at 10(-6) M to P at 10(-7) M (12.43 +/- 3.31%) or P at 10(-6) M (13.52 +/- 4.10%); however, this effect was not significantly different from P alone. Coincubation of P or RU486 with spermatozoa during capacitation did not stimulate the acrosome reaction in the concentrations tested. CONCLUSION: Progesterone directly stimulates human sperm HA transiently. Progesterone has no significant effect on acrosome reaction in capacitating spermatozoa. The effects of P are rapid and not counteracted by RU486, suggesting that the mechanism of action of P may not be mediated by specific P nuclear receptors.     

The sirtuin 1 activator YK 3-237 stimulates capacitation-related events in human spermatozoa

Research questionDoes sirtuin-1 (SIRT1) have a role in the human spermatozoa capacitation process?DesignHuman spermatozoa were incubated for 6 h in a capacitating medium in presence or absence of the specific SIRT1 activator, YK 3-237. Several sperm parameters were determined by flow cytometry: viability, acrosome reaction and mitochondria membrane status. Sperm motility was determined objectively by computer-assisted semen analysis. Sperm capacitation status was evaluated by the extent of protein tyrosine phosphorylation and by the percentage of spermatozoa with the acrosome reacted by a calcium ionophore challenge.ResultsSIRT1 was detected in the connecting piece of human spermatozoa where a lysine acetylation pattern was mainly found along the sperm tail. SIRT1 activation accelerates the occurrence of a phenotype associated with human sperm capacitation, with no differences seen in the lysine acetylation pattern. After 1 h of co-incubation of YK 3-237 with human spermatozoa, tyrosine phosphorylation levels were comparable to control levels after 6 h of incubation in capacitating conditions. In addition, the activator improved sperm responsiveness to a Ca²⁺ ionophore (A23187) challenge determined by an increase in acrosome-reacted spermatozoa (P = 0.025). Importantly, sperm viability and mitochondrial activity-related parameters assessed by flow cytometry were not affected by YK 3-237.ConclusionYK 3-237 induces capacitation-related events in human spermatozoa such an increase of tyrosine phosphorylation levels and acrosome-reacted spermatozoa after the ionophore challenge. Together, these results show that YK 3-237 affects human spermatozoa capacitation-related events by a mechanism independent of protein lysine acetylation but dependent on bicarbonate and calcium.     

The effects of prolactin on human sperm capacitation and acrosome reaction

OBJECTIVE: To study the effects of human prolactin (PRL) on human sperm capacitation and acrosome reaction. DESIGN: Acrosome reactions were induced by the addition of follicular fluid (FF) and progesterone (P). Experiments were performed to determine time and dose-dependent effects of PRL on sperm capacitation, potentiation of the acrosome reaction, decapacitating effects, and potential for PRL to induce an acrosome reaction. RESULTS: An average of 31.5% of spermatozoa underwent acrosome reaction with addition of FF and P. No time- or dose-dependent PRL effects on sperm capacitation or acrosome reaction were found (P greater than 0.05). CONCLUSION: Prolactin does not play an important role in human sperm capacitation or acrosome reaction.