考马期亮蓝染色法检测人精子顶体反应的评价

为建立一种检测人检测人精子顶体反应的简便实用的新技术，用３．５％高氧酸水溶液本制０．０５％考马斯亮蓝染色液浸染人精子３０ｍｉｎ，顶体完整者顶体区染成紫蓝色，顶体反应者则不梁。对获能前后和钙离子载体Ａ２３１８７诱导顶体反应的精子进行染色并与经典的顶体反应检测技术ＰＳＡ法对照，两种方法检测顶体反应率无显著差异。     

整合素系统对人精子顶体反应的作用

目的：观察整合素系统与人精子顶体反应的关系。方法：采用整合素VLA—5的特异性配体纤连蛋白(Fn)及整合素配体的活性片断RGD肽作用于人精子lh后，用三色法行顶体染色，采用了常规显微镜下肉眼计数和图像处理软件进行分析两种方法，观察Fn和RGD作用前后顶体反应(AR)率的变化。结果：常规显微镜下肉眼计数发现，100nM的Fn、200nM的Fn、250mg/L的RGD肽和500mg/L的RGD肽作用于人精子后AR率较作用前有明显增高；运用图像分析软件分析发现，100nW的Fn、200nM的Fn、250mg/L的RGD肽和500mg／L的RGD肽作用于人精子后AR率也有显性升高。以上P均＜0.05。结论：人精子膜上的整合素参与了人精子的顶体反应。     

宫颈粘液对人精子甘露糖受体表达的影响

目的：研究人宫颈粘液是否影响人精子甘露糖受体的表达。方法：正常人精子穿透宫颈粘液2h后．用金霉素（CTC）荧光染色法鉴定其获能及顶体状态,并用异硫氰酸荧光素标记的甘露糖化牛血清白蛋白（FITC-DMA）检测精子甘露糖受体的表达。对照组分别用获能培养基BWW培养0、2、6h后用相同方法检测。结果：人精子穿透宫颈粘液后“获能”型精子百分率较穿透前增高,而“顶体反应”型精子百分率及甘露糖受体的标记阳性率与穿透前无差别。结论：宫颈粘液促进精子获能,但不诱导顶体反应,且不影响其甘露糖受体的表达。     

荧光染色技术在精子检测中的应用

近20年来，随着荧光染色技术的不断发展完善，其在检测精子染色质、质膜完整性、线粒体活性、精子获能状态及顶体状态等方面取得成功。荧光染色技术利用荧光染色试剂能特异的与精子染色质、质膜、线粒体和顶体上某些成分结合，在激发光激发下能发出肉眼可见的荧光并在荧光显微镜下观测，从而迅速、准确地了解精子各方面的功能。该技术的应用有助于了解精子的受精能力，为选择助孕方式提供可靠依据，尤其对原因不明不孕症患者的诊断治疗，更见优势。目前多主张两种或三种方法联合使用，如：三重染色法同时检测精子质膜、线粒体活性和项体状态。     

豚鼠精子的膜领域和顶体反应的膜融合活动的作用

在本文,作者检测了豚鼠精子的顶体周围的浆膜(PM)和外顶体膜范围的结构,并当顶体反应的膜融合活动时,确定其结局。附睾尾精子排列成卷曲状,并有顶体周围浆膜“连结”区参与,在这些区域浆膜是由不全晶体腊梅糖构成的交叉桥所连结。在腹(凹)面桥物质把浆膜     

猕猴精子获能和顶体反应过程中Ca~（2＋）和Ca~（2＋）－ATPase定位及咖啡因和dbcAMP对顶体反应的影响

用焦锑酸钾原位沉淀法和枸橼酸铅捕获法研究了猕猴精子获能和顶体反应过程中的Ｃａ~（２＋）分布和Ｃａ~（２＋）－ＡＴＰａｓｅ活性。获能前精子头部Ｃａ~（２＋）主要分布于顶体区质膜内外、顶体外膜和顶体内膜内侧，尾部Ｃａ~（２＋）主要分布于线粒体基质，在质膜、致密纤维和轴丝处也有一定分布，在获能过程中Ｃａ~（２＋）进入精子内部，并在头部结合于顶体区质膜内侧和顶体外膜外侧。顶体反应时Ｃａ~（２＋）结合于顶体内膜外侧和由顶体外膜囊泡化形成的囊泡内外，另外还有一些Ｃａ~（２＋）分散存在于顶体内容物中。在ｐＨ９．０条件下，头部Ｃａ~（２＋）－ＡＴＰａｓｅ活性存在于顶体外膜外侧和顶体区质膜外侧，顶体后区质膜无此酶活性。尾部Ｃａ~（２＋）－ＡＴＰａｓｅ存在于质膜、线粒体膜及致密纤维和轴丝处。各处的Ｃａ~（２＋）－ＡＴＰａｓｅ活性在获能和顶体反应过程中一直存在。咖啡因和ｄｂｃＡＭＰ可提高精子运动能力，前者还可显著增加顶体反应率。     

人精子甘露糖受体表达与顶体反应的关系

目的 研究人精子的甘露糖受体(MR)表达与顶体反应的关系，以探讨体外获能培养精子的MR表达是否为人精子获能的标志。方法 将上游法收获的活精子在获能培养基BWW中进行体外获能培养后，加入小鼠透明带溶解液(mZPS)诱导人精子顶体反应，精子悬液用异硫氰酸荧光素标记的甘露糖基化牛血清白蛋白(FITCDMA)为探针标记MR，用豌豆凝集素(PSA)法检测顶体反应。结果 体外获能培养人精子的MR表达率与mZPS诱导的顶体反应率无相关性。结论 体外获能培养精子的MR表达可能不是人精子获能的标志。     

猕猴精子获能和顶体反应过程中Ca^2＋和Ca^2＋—ATPase定位及咖啡因…

用焦锑酸钾原位沉淀法和枸橼酸铅捕获研究了猕猴精子获能和顶体反应过程中的Ｃａ＾２＋分布和Ｃａ＾２＋－ＡＴＰａｓｅ活性。获能前精子头部Ｃａ＾２＋主要分布于顶体区质膜内外、顶体外膜和顶体内膜内侧，尾部Ｃａ＾２＋主要分布于线粒体基质，在质膜、致密纤维和轴丝处也有一定分布。在获能过程中Ｃａ＾２＋进入精子内部，并在头部结合于顶体区质膜内侧和顶体外膜外侧。顶体反应时Ｃａ＾２＋结合于顶体内膜外侧和由顶体外膜囊泡化     

甲氧滴滴涕对大鼠精子顶体反应的影响

目的:探讨甲氧滴滴涕(methoxychlor,MXC)对雄性大鼠精子顶体反应的影响。方法:在体外培养的精子悬液中加入不同浓度的MXC和孕酮处理,通过精子考马斯亮蓝染色,镜检计数顶体反应型精子的百分率。结果:与空白组比较,单纯经MXC处理组的顶体反应型精子百分率明显增加,另加孕酮的各处理组顶体反应型精子的百分率增加尤为明显,MXC终浓度≤6.25μg/ml的处理组顶体反应型精子百分率不增加。结论:MXC可促进大鼠精子发生顶体反应且与其剂量有关,当MXC终浓度≤6.25μg/ml不诱发顶体反应。MXC能加强孕酮诱发精子顶体反应的作用。     

牛精子头后部质膜分区的研究

检测牛精子头 存在的不同质膜区域。方法先用钙离子载体Ａ２３１８７诱导精子发生顶体反应,然后用水浴超声波和低速离心法分离得到纯牛精子头。直接用顶体反应后精子和低渗处理的顶体反应后精子头免疫Ｂａｌｂ／ｃ母鼠和新西兰公兔,制备抗牛精子抗体,     

Human testicular protein TPX1/CRISP-2: localization in spermatozoa, fate after capacitation and relevance for gamete interaction

Testicular protein Tpx-1, also known as CRISP-2, is a cysteine-rich secretory protein specifically expressed in the male reproductive tract. Since the information available on the human protein is limited to the identification and expression of its gene, in this work we have studied the presence and localization of human Tpx-1 (TPX1) in sperm, its fate after capacitation and acrosome reaction (AR), and its possible involvement in gamete interaction. Indirect immunofluorescence studies revealed the absence of significant staining in live or fixed non-permeabilized sperm, in contrast to a clear labelling in the acrosomal region of permeabilized sperm. These results, together with complementary evidence from protein extraction procedures strongly support that TPX1 would be mainly an intra-acrosomal protein in fresh sperm. After in vitro capacitation and ionophore-induced AR, TPX1 remained associated with the equatorial segment of the acrosome. The lack of differences in the electrophoretic mobility of TPX1 before and after capacitation and AR indicates that the protein would not undergo proteolytical modifications during these processes. The possible involvement of TPX1 in gamete interaction was evaluated by the hamster oocyte penetration test. The presence of anti-TPX1 during gamete co-incubation produced a significant and dose-dependent inhibition in the percentage of penetrated zona-free hamster oocytes without affecting sperm motility, the AR or sperm binding to the oolema. Together, these results indicate that human TPX1 would be a component of the sperm acrosome that remains associated with sperm after capacitation and AR, and is relevant for sperm-oocyte interaction.     

Effect of sperm-antibodies on acrosome reaction of human sperm used for the hamster egg penetration assay

The effect of anti-sperm antibodies (ASA) on the rate of acrosome reactions (AR) during "in vitro" capacitation of human sperm used for the hamster egg penetration assay (HEPA) was assessed. Motile sperm suspensions from donors were exposed to several sera and seminal plasma with sperm head-directed ASA, then they were washed and capacitated "in vitro." After capacitation, the proportion of acrosome-reacted viable sperm was assessed by staining with Fluoresceinated Pisum Sativum Agglutinin and supravital stain Hoechst 33258. ASA of any immunoglobulin class did not significantly affect either the AR rate, or the hamster egg penetration rate. In conclusion, interference of ASA on spontaneous AR rate during "in vitro" capacitation can not be advocated as an explanation of the impairment of the interaction of human sperm with egg or its vestments, which have been reported in several studies.     

An anti-actin monoclonal antibody inhibits the zona pellucida-induced acrosome reaction and hyperactivated motility of human sperm.

We report an inhibitory effect of an anti-actin monoclonal antibody (mAb) on the human zona pellucida (ZP)-induced acrosome reaction (AR). Motile sperm were incubated with native human ZP for 2 h in medium containing either the anti-actin mAb, an irrelevant control mAb or cytochalasins B or D (40 micromol/l). Sperm bound to the ZP were recovered and the AR was determined by fluorescein-labelled Pisum Sativum agglutinin. Anti-mouse immunoglobulin G (mIgG) Dynabeads, immunofluorescence and immunogold were used to detect the location of the anti-actin mAb in sperm. The anti-actin mAb significantly inhibited the ZP-induced AR (equivalent to cytochalasins), the ionophore A23187-induced AR and hyperactivation of sperm in medium. After incubation with anti-actin mAb, anti-mIgG beads bound to the head of >50% of sperm recovered after binding to the ZP and 10% of sperm remaining in the medium. The proportion of sperm that bound anti-mIgG beads after recovery from binding to the ZP in the presence of the anti-actin mAb was significantly correlated with the ZP-induced AR in the absence of the antibody. Immunofluorescence and immunogold demonstrated entry of the anti-actin mAb into sperm. This study suggests that the sperm plasma membrane becomes permeable to the anti-actin mAb during capacitation and initiation of the AR.     

An important role of actin polymerization in the human zona pellucida-induced acrosome reaction.

The effects of inhibitors of actin polymerization and depolymerization, cytochalasins and phalloidin, on the human zona pellucida (ZP)-induced acrosome reaction (AR) were investigated. Motile spermatozoa, selected by swim-up technique from normozoospermic men, were incubated in medium with or without the actin modulators. Oocytes (four per test) which had failed to fertilize in vitro were added and incubation continued for 2 h. The spermatozoa bound to the ZP were dislodged by repeatedly aspirating the oocytes with a small-bore pipette and the status of the acrosomes was determined by fluorescein-labelled Pisum sativum agglutinin (PSA). Double immunofluorescent staining with PSA and an anti-actin monoclonal antibody illuminated the acrosomal region of acrosome-intact spermatozoa. In calcium ionophore-induced AR spermatozoa, actin staining was confined to the equatorial segment, post-acrosomal region and tail. Cytochalasins B and D significantly inhibited ZP-induced AR in a dose-dependent manner (P < 0.001). Both inhibitors had no effect on the acrosome of spermatozoa in the insemination medium. Cytochalasin B or D (10-40 micromol/l) had no effect on total percentage motile spermatozoa but decreased sperm velocity and hyperactivation. Phalloidin had no effect on the ZP-induced AR or sperm motility. In conclusion, actin polymerization plays an important role in human ZP-induced AR.     

Human sperm capacitation and the acrosome reaction.

A model is presented that describes the mechanism of human sperm capacitation and the acrosome reaction. The processes of capacitation and the acrosome reaction are proposed to function in control of the activation/release of acrosomal enzyme(s) involved in sperm penetration through the zona pellucida. During capacitation, the sperm head membranes are biochemically modified, allowing the acrosome reaction to take place when the spermatozoon approaches or reaches the zona pellucida, resulting in the localized activation and release of the appropriate enzyme(s). Further, capacitation is presented as a continuing process that occurs during sperm transport through the female genital tract and is physiologically not completed until the spermatozoon reaches the oocyte (unless the spermatozoa are kept at a particular genital tract site for prolonged periods). The biochemical alterations that occur during capacitation are discussed. It is suggested that extensive modifications in the lipid bilayer structure, e.g. in the cholesterol or phospholipid content, are not part of capacitation because such changes would prematurely destabilize the membranes. Rather, such changes occur during the acrosome reaction. It is also proposed that the human sperm acrosome reaction has many similarities to the somatic cell exocytotic events which occur during the regulated pathway of secretion. One or more oocyte stimuli result in the activation of protein kinases, likely (but not necessarily) via activation of G-protein coupled receptors on the sperm plasma membrane and the formation of second messengers. The kinases phosphorylate and activate proteins, continuing the biochemical cascade that ultimately results in the acrosome reaction. The role of other enzyme systems such as those involved in ion transport, proteolysis, phospholipid metabolism (including that of arachidonic acid) and other metabolic events, is discussed. Calcium ion influx as initiator of the acrosome reaction is reconsidered. The proposed model also takes into consideration the structural events of membrane fusion.     

Detection of human sperm acrosome reaction: comparison between methods using double staining, Pisum sativum agglutinin, concanavalin A and transmission electron microscopy

The acrosome reaction is an important marker for human sperm function. Since different laboratory techniques may be used for the detection of this exocytotic process, the purpose of the present study was to compare three common markers [Pisum sativum agglutinin (PSA), concanavalin A (ConA), double staining] and transmission electron microscopy for identification of acrosomal changes. Preliminary findings had demonstrated that similar results were achieved with Trypan Blue and Hoechst 33258 staining. Therefore, supravital stainings were omitted. In various experiments, human spermatozoa were treated with two concentrations (10 and 3.3 microM) of calcium ionophore A23187 for 15, 30 and 60 min after capacitation for 3 and 6 h at 37 degrees C. The percentages of spermatozoa with acrosomal loss detected by fluorescein isothiocyanate (FITC)-ConA were consistently lower than those obtained by double staining or FITC-PSA, which showed comparable results. Following 6 h of capacitation and incubation with 10 microM ionophore for 1 h at 37 degrees C, 25.9 +/- 15.7% of all spermatozoa showed almost complete loss of the acrosomal content. Binding of FITC- ConA to the acrosomal region was observed in 27.0 +/- 13.2% of spermatozoa obtained from the same sample. FITC-ConA and double staining or FITC-PSA detect different stages of the acrosome reaction and may be helpful for a differentiated evaluation of this sperm function.      

In vitro sperm capacitation to treat antisperm antibodies bound to the sperm surface.

Our objective was to study antisperm antibody bound to the acrosome region during in vitro capacitation and to determine whether acrosome-antibody free sperm can be obtained from previously acrosome-antibody-coated sperm. The spermatozoa from a selected series of 14 patients were tested for sperm antibodies bound to the sperm surface using d-IBT and focusing on the acrosome positivity. The tests were carried out before the incubation and after 3, 6, 9, and 12 h of incubation in Tyrode's solution with 0.5% human serum albumin as the capacitation medium. Tests to evaluate acrosome region, sperm motion parameters, and zonae binding ability were carried out. In this way we were able to evaluate sperm function during capacitation protocol. The patients were 14 subjects selected according to good seminal characteristics, good post-rise sperm parameters, and high percentage of ASA bound to the sperm surface. In all cases the results showed that antisperm antibodies bound to the acrosome region were shed prior to the acrosome reaction. During sperm capacitation in human a modification, migration, or shedding of plasma membrane molecules takes place. The presence of antibodies in such an important area of the sperm head could certainly interfere in the fertilization process. Our data indicate that in vitro capacitation could provide an in vitro therapy capable of eluting antibodies from the acrosome region.     

Pentoxifylline-stimulated capacitation and acrosome reaction in hamster spermatozoa: involvement of intracellular signalling molecules.

We investigated the role of cAMP/cGMP, protein kinases and intracellular calcium ( [Ca2+]i) in pentoxifylline-stimulated hamster sperm capacitation and the acrosome reaction (AR) in vitro. Treatment with pentoxifylline (0.45 mM) initially increased sperm cAMP values 2.8-fold, compared with untreated controls (396 +/- 9.2 versus 141 +/- 6.0 fmoles/10(6) spermatozoa; mean +/- SEM, n = 6) after 15 min, although by 3 h, cAMP values were similar (503-531 fmoles/10(6) spermatozoa). cGMP values ( approximately 27 fmoles/10(6) spermatozoa) were the same in treated and control spermatozoa. Both sperm capacitation and the AR, determined from the absence of an acrosomal cap, were stimulated by pentoxifylline; these were almost completely inhibited by a Cl-/ HCO3- antiporter inhibitor (4,4-diisothiocyanato-stilbene-2,2 disulphonic acid; 1 mM) defined from the degree of sperm motility and by a protein kinase A inhibitor (H89; 10 microM). A protein kinase C inhibitor (staurosporine, 1 nM) did not affect pentoxifylline-stimulated capacitation but inhibited the AR by 50%. A protein tyrosine kinase inhibitor (tyrphostin A-47, 0.1 mM) had no effect on either pentoxifylline-stimulated capacitation or AR. A phospholipase A2 inhibitor (aristolochic acid, 0.4 mM) markedly inhibited the pentoxifylline-stimulated AR but not capacitation. When intracellular sperm calcium [Ca2+/-]i was measured using fura-2-AM, there was an early rise (271 nM at 0.5 h) in pentoxifylline-treated spermatozoa; this appeared to be due to intracellular mobilization rather than to uptake. In the absence of extracellular Ca2+, sperm motility was maintained in the presence of pentoxifylline, but capacitation did not occur; spermatozoa exhibited a low level of hyperactivated motility and had a poor rate of AR (20.5 +/- 2.3%). These results suggest that: (i) the pentoxifylline-stimulated early onset of sperm capacitation may be mediated by an early rise in cAMP and [Ca2+/-]i and involves protein kinase A activity; and (ii) pentoxifylline-stimulated AR may require phospholipase A2 and protein kinase C activity.     

Assessment of Progesterone-Induced Acrosome Reaction by Biotinylated Monoclonal Antibody Probes

PROBLEM: To develop an immunohistochemical assay for determination of acrosome-reacted human sperm and to study the effects of progesterone and cholesterol treatment on human sperm acrosome reaction. METHOD OF STUDY: Three distinct anti-sperm monoclonal antibodies were biotinylated and used as probes for assessment of acrosome reaction in a 30-min immunohistochemical assay. Progesterone and/or cholesterol were added to sperm preparation to influence the acrosome reaction in different experimental conditions. RESULTS: Percentages of acrosome-intact sperm decreased significantly during the 18-hr incubation. Acrosome reaction could be induced by progesterone as early as 2 hr after sperm incubation in human tubal fluid. The degree of progesterone-induced acrosome reaction was time dependent and the optimal effect was reached by adding 10 μg/ml progesterone for 30 min incubation. Progesterone-induced acrosome reaction was shown to be hormone-concentration dependent with 50% stimulation at 1 μg/ml. Cholesterol (1 μg/ml) was found to inhibit progesterone-induced acrosome reaction either by co-incubation with human sperm during capacitation, or by simultaneous incubation with progesterone during acrosome reaction induction. CONCLUSIONS: Assessment of human sperm acrosomal status by avidin-biotin immunohistochemical assay can be a routine in clinical laboratories for male infertility services. Cholesterol can inhibit progesterone-induced acrosome reaction, possibly by its modifications of sperm plasma membrane and/or interference of progesterone binding to its surface receptors.