跨膜移动率:一种简便客观的精子运动力检测方法

本实验应用精子跨膜移动率(TMMR)测定方法,考察了光镜下精子活动率与TMMR的相关性。同时还用杀精剂壬苯醇醚,精子获能液BWW以及精子运动激动剂咖啡因分别作用于射出精子,观察精子TMMR的改变。结果显示精子TMMR与光镜下估测的精子活动率有高度相关性(r=0.64,p<0.005)。精子的TMMR随壬苯醇醚作用浓度的增加而下降;BWW和咖啡因都能使精子活动力增加,并使精子TMMR明显上升。本实验方法简便,操作容易,数据客观,具有明显的可行性和实用性。     

米非司酮的体外杀精作用及其机理探讨

米非司酮的体外杀精作用及其机理探讨王春年，褚劲松，谢文英本实验应用钙离子的荧光指示剂Ｆｕｒａ－２ＡＭ和计算机辅助的图象分析技术，对米非司酮（ＲＵ４８６）的体外杀精最大稀释度及其对精子内游离钙离子含量的影响进行了研究。一、材料和方法１．精液标本的采集及...     

输精管结扎术时精囊灌注消毒剂杀精效果的比较研究

应用消毒剂新洁尔灭、洗必泰醋酸盐,利凡诺作体外杀精子试验,其最低杀精浓度分别为0.25mg/ml、2.5mg/ml、7.5mg/ml。采用三种药物以最低杀精浓度总量8、10、14和16ml分别进行输精管结扎术时精囊灌注207例。临床观察表明:从精囊灌注后第一次尿中可排出精子3.6～60×10~6个,尿中精子总数在不同灌注量之间差异不明显,各药物组与生理盐水对照组无差异。术后第一次排精和术后1月取精液作常规检查者194例,未发现精子者分别为101例(52％)和169例(87.1％),其余虽有精子,但数量均在4×10~6/ml以内,并且三种药物分别灌注组的精子活力均完全消失。     

性激素对化疗患者睾丸生精功能的保护及机理

在恶性肿瘤和一些自身免疫性疾病的治疗中 ,化疗是必不可少的治疗措施 ,但研究发现化疗药物对男性睾丸生精功能损害极大 ,可部分或全部杀死各级生精细胞 ,使男性精子数量减少和 /或精子活力降低 ,甚至不育。研究发现在化疗的不同时期应用不同的性激素可保护睾丸生精功能。近年来对睾酮和雌激素、促性腺激素释放激素类似物或激动剂、雄激素拮抗剂、促性腺激素释放激素 拮抗剂在化疗中对睾丸生精功能的保护及机理进行了较多的研究 ,并取得一定的成绩。     

小鼠腹腔注射环磷酰胺建立少精子症模型的稳定性探讨

近来有人寻求用腹腔注射环磷酰胺的方法诱导小鼠以建立一种经济、周期短、便于获取的少精子症模型并用于实验研究。已有研究证实环磷酰胺可诱导小鼠睾丸生精细胞大量凋亡、精子数量减少。但小鼠生精上皮再生速度很快，诱导后能否在相当长时间内仍保持显著低的生精水平，抑或有一个快速恢复过程，即模型的稳定性问题，目前尚无结论，因此在此平台上进行下一步的治疗性实验并不一定可靠。本实验对此进行研究，并初步探讨其作用机制。     

AF-2364，一种杀精子剂候选成分

通过靶向作用于精子线粒体，抑制精子运动进行避孕是一种作用特异的、很有前景的避孕方式。AF-2364，是氯尼达明（Londidamine，LND）的类似物，其毒副作用显著降低，目前被认为是一种很有潜力的男性避孕的候选化合物。LND亦可作用于肿瘤细胞线粒体抑制能量代谢。目前尚无AF-2364对于人类精子功能作用的相关报道。在本研究中，我们探寻了AF-2364的体外杀精子作用及其初步机制。体外实验显示，AF-2364显著抑制人类精子的运动：进一步的研究发现，AF-2364可作用于精子线粒体膜通透性转换（PT）孔，降低线粒体膜电位（mitochondrial membrane potential，△ψm），抑制线粒体能量代谢，使ATP下降，从而使精子制动。我们同时检测了AF-2364作用后细胞骨架一系列蛋白调控通路在人精子中的变化及人精子蛋白组学改变，未见显著差异；AF-2364与人及小鼠其他细胞系孵育实验提示较低浓度下的AF-2364对人精子制动作用的特异性。综上，AF-2364可通过对精子线粒体PT孔的直接作用使精子制动，并可能发展为一种杀精子剂的候选成分。     

五苦栓杀精抑菌作用的实验研究

目的 :了解中药“五苦栓”杀精、抑菌作用。　方法 :运用中药五倍子、苦参粗提物配制成杀精栓剂 (WK) ,对大鼠精子行杀精、生育、阴道粘膜刺激及体外抑菌试验。　结果 :WK具有较明显杀精作用 ,其体外杀精效果在高浓度组超过NP 9阳性对照组 (P <0 .0 1) ,中等浓度时与NP 9无显著性差异 (P >0 .0 5 )。体内杀精效果与NP 9无差异 (P >0 .0 5 )。对淋病奈瑟菌及霉菌的抑制作用强于NP 9(P <0 .0 1) ,且安全性实验未发现有明显副作用。　结论 :“五苦栓”杀精及体外抑制淋病奈瑟菌、霉菌作用确切 ,具有临床开发价值。     

中药还精方对人精子运动及相关参数的影响

本研究采用血清药理学的方法,通过人精子与含药血清共培养,观察了中药还精方对人精子运动的作用,并运用计算机辅助的精液自动分析系统客观地评价中药还精方对精子的影响,以期进一步阐明中药还精方在男性不育症治疗中的作用.     

精原干细胞去分化和多能性的研究进展

精原干细胞是具有终身有丝分裂能力进行自我复制同时也可分化成单倍体精子的生殖细胞。最近,一些研究发现精原干细胞可在体外被诱导成多能干细胞。提示精原干细胞可成为再生医学所需多能干细胞的很好来源,尤其是获得没有免疫排斥、患者自身来源的多能干细胞。本综述将重点介绍目前对精原干细胞多能性的认识及其在再生医学中应用的优势。     

精子上游处理技术的改进和影响因素的评价

本文介绍一种改进的精子上游处理技术,用以提高活动精子的回收率,并对其影响因素进行了分析。共选择精子密度正常的精子活力低下症22例进行该上游处理,活动精子回收率为50.07±23.4％。研究发现不同精子密度、精子运动速度、精液粘稠度等因素可影响活动精子回收率。作者认为该法设备操作简单,可复性强,便于在人工授精中推广应用。     

腺苷脱氨酶在人精子膜上的定位及其单克隆抗体对人精子穿卵能力的影响

利用本实验室制备的抗人精子膜结合腺苷脱氨酶（ＡＤＡ）单克隆抗体（ＭｃＡｂ）和间接荧光免疫技术，分析了该酶在正常人精子膜上的位置。同时，利用该酶单抗分析了该酶活性对人精子穿透去透明带卵的影响。上述两个实验的结果显示：（１）ＡＤＡ结合于人精子中段、尾部膜的外侧；（２）利用抗ＡＤＡＭｃＡｂ抑制ＡＤＡ活性可明显抑制正常人精子穿透去透明带卵的能力。这一研究结果提示，ＡＤＡ作为精子的膜蛋白组分，可能参与对精子生理功能的调节过程。     

Effect of ace-inhibitors, calmodulin antagonists, acetylcholine receptor blocking, and alpha receptor blocking agents on motility of human sperm

The purpose of this study was to investigate the influence of several pharmacological compounds on the motility and velocity of washed human spermatozoa. Results were evaluated by multiple exposure photography and computer-aided picture analysis. The motility-inhibiting effect of the antifertility drug gossypol was confirmed. Gossypol proved to be a potent inhibitor of the angiotensin converting enzyme (ACE) detectable in high concentrations in seminal plasma. However, human sperm motility was not inhibited during incubation with two other specific ACE-inhibitors (captopril, enalapril). On the contrary, high concentrations of captopril even showed a slight motility-stimulating effect. These results indicate no direct involvement of ACE in the regulation of sperm motility but suggest a direct interaction of gossypol with the plasma membrane of spermatozoa. To clarify whether or not gossypol blocks membranous ion transport, the effect of well-defined ion transport blocking agents on sperm motility was investigated. It was determined that the acetylcholine receptor blocker alpha-bungarotoxin and trifluoperazine, a specific calmodulin antagonist, inhibit sperm motility completely. Since stimulation of sperm motility by captopril may be due to an alpha-mimetic action of this compound, the influence of two alpha receptor blockers (bromocriptine, lisuride) on sperm motility was studied. Although lisuride inhibited sperm motility completely, bromocriptine revealed no influence. A temporary and reversible intervention with membrane transport processes could be a suitable way to regulate human sperm motility and male fertility.     

Effect of progesterone on acrosome reaction, hypoosmotic swelling test, and DNA stability in human spermatozoa

The association between different sperm parameters, an in vitro effect of progesterone, has not been studied satisfactorily. In this article, the effect of progesterone on acrosome reaction (AR), plasma membrane integrity, and chromatin stability has been assessed in human spermatozoa with normal morphology and motility. Semen samples were obtained by masturbation from 25 patients. Two criteria of classification were utilized in this study: high motility group and normal morphology group incubated with progesterone. The effect of progesterone on AR, plasma membrane integrity, and chromatin stability in human spermatozoa with normal morphology and motility was realized. The results suggest that only the subpopulation of spermatozoa with normal morphology is able to undergo the progesterone-induced AR. It is possible that in the reproductive female tract it takes place a high selection of sperm with chromatin stability determined and optimal plasma membrane to undergo the AR prerequisite for the fecundation.     

Interaction of PDC-109, the major secretory protein from bull seminal vesicles, with bovine sperm membrane Ca2+-ATPase

PDC-109 is the prevalent secretory protein from bovine seminal vesicles that binds to the midpiece of sperm once they pass the ampulla of the vas deferens during emission. Thereby, the protein changes biophysical membrane properties, eventually resulting in increased sperm motility. To elucidate the underlying biochemical mechanism, we have studied the ion-pumping activity (Ca(2+)-ATPase) in membrane preparations of bovine spermatozoa following in vitro incubation with the protein and analyzed whether PDC-109 influences sperm motility. PDC-109 was purified to homogeneity from bull seminal vesicle extracts using a newly described method. The effect of PDC-109 on sperm motility was analyzed using the CASA-method. These experiments clearly demonstrated that PDC-109 significantly increases sperm motility. Calcium-pumping mechanisms were analyzed by monitoring the effect of PDC-109 on various parameters of enzyme activity of Ca(2+)-ATPase in epididymal sperm plasma membranes and were compared with Ca(2+)-ATPase activities from other organs and from epididymal sperm of different species, respectively. Specificity studies were performed using different Ca(2+)-antagonists. Enzyme activities of both Mg(2+)-dependent and Mg(2+)-independent Ca(2+)-ATPases increased in a dose-dependent manner following the addition of the PDC-109 (range 5-20 microg). Preincubation of PDC-109 at temperatures above 37 degrees C and pHs ranging from below 6.5 and above 8.5 led to the loss of the stimulatory effect. An analysis of enzyme kinetics pointed to irreversible, cooperative interaction of PDC-109 with the enzyme. The effect was organ-specific, that is, restricted to sperm ATPases, but it was not species-specific, as it could be elicited also in rat sperm.     

The mitochondria of stallion spermatozoa are more sensitive than the plasmalemma to osmotic-induced stress: role of c-Jun N-terminal kinase (JNK) pathway

Cryopreservation introduces extreme temperature and osmolality changes that impart lethal and sublethal effects on spermatozoa. Additionally, there is evidence that the osmotic stress induced by cryopreservation causes oxidative stress to spermatozoa. The main sources of reactive oxygen species in mammalian sperm are the mitochondria. In view of this, the aim of our study was to test whether or not osmotic stress was able to induce mitochondrial damage and to explore the osmotic tolerance of the mitochondria of stallion spermatozoa. Ejaculates from 7 stallions were subjected to osmolalities ranging from 75 to 1500 mOsm/kg, and the effect on sperm membrane integrity and mitochondrial membrane potential was studied. Additionally, the effects of changes in osmolality from hyposmotic to isosmotic and from hyperosmotic to isosmotic solutions were studied (osmotic excursions). The cellular volume of stallion spermatozoa under isosmotic conditions was 20.4 ± 0.33 μm(3). When exposed to low osmolality, the stallion spermatozoa behaved like a linear osmometer, whereas exposure to high osmolalities up to 900 mOsm/kg resulted in decreased sperm volume. Although sperm membranes were relatively resistant to changes in osmolality, mitochondrial membrane potential decreased when osmolalities were low or very high (10.7 ± 1.74 and 16.5 ± 1.70 at 75 and 150 mOsm/kg, respectively, and 13.1 ± 1.83 at 1500 mOsm/kg), whereas in isosmolar controls the percentage of stallion sperm mitochondria with a high membrane potential was 41.1 ± 1.69 (P < .01). Osmotic excursions induced greater damage than exposure of spermatozoa to a given nonphysiologic osmolality, and again the mitochondria were more prone to damage induced by osmotic excursions than was the sperm plasma membrane. In search of intracellular components that could mediate these changes, we have detected for the first time the c-Jun N-terminal kinase 1/2 in stallion spermatozoa, which are apparently involved in the regulation of the viability of these cells.     

Does the hypoosmotic swelling test predict human sperm viability?

Human sperm viability is essential for successful fertilization. Eosin Y is the usually accepted method for sperm viability assessment, though the hypoosmotic swelling test has been proposed for the selection of viable spermatozoa in procedures such as intracytoplasmic sperm injection. The present study was designed to determine the value of hypoosmotic swelling test in the prediction of sperm viability. For this purpose, hypoosmotic swelling and eosin Y were performed in parallel and in combination, on both fresh and freeze-thawed semen. Rates for eosin Y were significantly higher than for the hypoosmotic swelling test in fresh semen, with a weak, though significant correlation between the two tests (r = 0.47, p < 0.05). When both tests were performed in succession (hypoosmotic swelling test followed by eosin Y), 14.6% of swollen sperm incorporated the dye. Following exposure to hypoosmotic conditions, sperm viability decreased by 35%. When sperm were killed by freezing, hypoosmotic swelling test rates were higher than eosin Y. Results indicate that these two tests cannot be used interchangeably, since 15% of the swollen sperm apparently died, suggesting that plasma membrane integrity is lost before the capacity to maintain osmotic equilibrium.     

Clusterin在人精子膜表面的表达及其意义

目的 探讨clusterin在人类精子中的表达及其意义.方法 从精子表面用1%TritonX-100提取及氯仿/甲醇分离疏水性的膜表面蛋白,用免疫印迹法检测精子表面是否存在大然clusterin蛋白.用免疫荧光方法确定clusterin在人精子膜表面的分布特点.结果 人精子膜表面及精浆中含有大量的clusterin,且clusterin仅分布于精子头部及顶体区域,而阴性对照组没有发现天然clusterin的存在.结论 人精子头部及顶体表面分布大量的clusterin,可能与精子成熟及精卵结合有关.     

Heparin-glutathione III: study with fluorescent probes as indicators of membrane status of bull sperm

Sperm obtained from bull epididymes were used to validate in vitro the effect of heparin and reduced glutathione on sperm membrane status, with the use of sodium dodecyl sulfate (SDS) and Triton X-100 in the presence of propidium iodide (IP) and diacetate fluorescein (FDA). The metabolic activities of treated sperm were qualitatively monitored using an alamar Blue Redox fluorescence indicator. Heparin did not damage the sperm plasma membrane, whereas GSH and SDS at 26 h of incubation dissolved the plasma membrane and the acrosome. On the other hand, at time zero, Triton X-100 showed 75% of sperm stained with IP, indicating plasma membrane damage. Results validated by electron microscopy of thin sections of treated sperm showed complete lack of the membrane, acrosome, and postacrosomal membrane system with 0.01% Triton X-100. Extracellular 15 mM GSH completely disappeared the plasma membrane over the sperm nucleus, leaving the postacrosomal membrane system and nucleus without apparent damage. The metabolic activity was supported over 52 h of incubation in any of the incubation systems tested, including Triton X-100, which showed a spermaticide effect. The authors propose that membrane damage does not mean they are dead, no matter the vital stain employed, and also that FDA-IP staining can be used as a fluorescent marker of sperm plasmatic membrane permeabilization and nuclear swelling.     

Sperm washing method alters the ability of seminal plasma proteins to revert the cold-shock damage on ram sperm membrane

Prolonged exposure of spermatozoa to seminal plasma (SP) has been found to have adverse effects on sperm function. Therefore, the separation of mammalian spermatozoa from SP is a practice routinely used in the laboratory and in assisted reproductive technology application. We have previously shown that adsorption of seminal plasma proteins (SPP) to the sperm cell surface partially restores the functional characteristics of damaged spermatozoa, reproducing those of live cells. In the present report, we have compared the influence of two different semen washing methods, a dextran/swim-up or a filtration procedure, on the SPP adsorption to the sperm surface, and on their ability to recover membrane integrity of cold-shocked sperm. Seminal plasma proteins were added to cold-shocked sperm samples obtained by both washing procedures. Adsorption of proteins to the sperm membrane surface was assessed by centrifugal counter-current distribution (CCCD) in an aqueous two-phase system, as well as membrane integrity being determined by fluorescence markers. The percentage of reversion of cold-shock effect in the sample containing plasma proteins with respect to the control sample was determined by assessing the percentage of membrane-intact spermatozoa, i.e. propidium iodide-negative. The addition of 700 microg of SPP to the swim-up damaged samples caused a 32% reversion, whereas the reversal percentage found with the same amount of SPP on filtered damaged samples was 10% (p < 0.05). Likewise, the loss of heterogeneity and the decrease in viability after the cold-shock revealed by CCCD analysis were greatly reversed by the addition of SPP to sperm samples obtained by swim-up. However, this restorative effect was again much lower when SPP were added to the cold-shocked filtered sample. These results strongly suggest that the washing method influences the ability of SPP to recover membrane integrity of cold-shocked sperm. Separation of spermatozoa by swim-up accounts for higher viability recovery than that obtained by filtration washing.     

精子膜蛋白研究进展

精子膜表面的蛋白是精子发生、成熟和精卵相互作用的重要分子,对其深入研究有助于揭示精子发生、成熟以及受精的分子机制。近年来,各种现代分子生物学技术以及生物信息学方法在生殖生物学领域的广泛应用,不仅使以前发现的一些精子膜蛋白相继得到克隆和测序,同时新的精子膜相关蛋白也不断被发现,为从基因和蛋白质水平研究其生物学功能、揭示生物生殖的分子机制并进而为避孕疫苗的开发等奠定了基础。本文就近年来精子膜蛋白的研究进展作一综述。