人精子染色体的制备方法

目的用人精子对去除透明带的金黄地鼠卵进行穿透,制备人精子染色体.方法将金黄地鼠行超排卵,取得金黄地鼠卵子进行去透明带处理,与体外处理并获能的人精子放子在一起受精,当形成原核或精子膨大头时,加入鬼臼毒素和长春新碱阻止原核融合和纺锤丝形成,经低渗、固定、老化等过程,获得人精子单倍染色体显带核型.结果与结论用此法检测2例有正常生育能力者精液,精子染色体分裂相分散的好.     

体外精子穿卵试验改进

目的将复杂的人精子体外穿卵试验进行改进和完善,使之利于常规开展.方法将金黄地鼠行超排卵,并将取得的金黄地鼠卵子进行去透明带处理,与体外处理并获能的人精子放在一起受精,通过低渗、固定染色制片,观察人精子穿透率.结果用该法检测了10例有正常生育能力精子穿透率,范围在30%～80%;20例精液常规正常原发不育者精子穿透率在10%以下.结论改进制片后穿卵试验是用于检测人精子受精能力一种方法.     

人精子表面整合素亚基α5、β1参与受精过程的研究

目的：研究人精子表面整合素亚基α5、β1在受精过程中的作用。方法：以人精子穿透去透明带金黄地鼠卵异种体外受精试验（SPA）作为检测人精子受精力的手段；分别用抗整合素亚基α5、β1抗体衣整合素特异的配体RGD肽作用于精子后，观察其受精力的改变。结果：用抗整合素亚基α5、β1抗体及RGD肽作用于精子后，受精率、受精指数及粘附指数均下降。结论：人精子表面整合素亚基α5、β1参与受精过程。     

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。     

HFMC在体外对精子功能和结构的影响

体外实验发现:HFMC能够全面抑制人精子的运动能力、穿透牛宫颈粘液的能力和穿透去透明带金黄地鼠卵的能力,随HFMC剂量增加或HFMC与精子作用时间延长,抑制作用增强;HFMC主要损伤精子的膜性结构。推测HFMC的这些作用可能是它在体内抗生育的机理之一。采用改良Sander-Cramer方法测得HFMC的体外最低有效杀精子浓度为10～22毫克/毫升,但对不同动物或同一动物不同部位精子的最低有效杀精子浓度不同。提示在研究避孕药物对精子的作用时,最好选用人精液精子或生物学特性与其相似、且来源容易的其他动物的精子。     

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。     

水凝胶HFMC的Ames试验研究(摘要)

水凝胶HFMC是一种以甲基丙烯酸羟乙酯为主要成份,并含有甲基丙烯酸、甲基丙烯酸乙酯的高分子聚合物。该剂具有全面抑制人精子的运动、穿透牛宫颈粘液和穿透去透明带金黄地鼠卵的能力。将水凝胶注入雄性动物的     

高分子聚合物水凝胶HFMC对人精子染色体的遗传性的研究

高分子聚合物水凝胶HFMC用作输精管节育材料因其具有可通透和自然复孕两大特性而有着广阔的应用前景。为了检测HFMC是否具有潜在的遗传毒性,本研究应用人精子染色体离体测试系统,在体外用经HFMC处理后的人精子与去透明带金黄地鼠卵受精,然后分析这些精子的染色体核型。结果表明:HFMC各剂量组与阴     

重组人卵透明带蛋白及其多克隆抗体对精子-透明带结合的影响

为了探讨毕赤酵母表达的重组人卵透明带ZP3蛋白(recombinant human zona pellucida-3 protein,rhZP3)及其多克隆抗体对小鼠和人精卵结合的影响,采用不同浓度的rhZP3以及空白培养液分别处理小鼠精子,然后再与小鼠卵子进行结合实验,观察经过不同处理的精子对透明带黏附及体外受精率的影响;用rhZP3以及空白培养液分别处理人精子,然后再与人卵子进行结合实验,观察经过不同处理的精子对透明带黏附的影响;用抗rhZP3抗体与阴性血清分别处理小鼠和人卵子,再与精子进行结合实验,观察多克隆抗体对精子粘附以及小鼠体外受精率的影响。实验结果表明,rhZP3和抗rhZP3多克隆抗体既能抑制人的体外精卵结合,也能抑制小鼠体外精卵结合,提示rhZP3具有天然透明带的特性,有发展成避孕疫苗和作为检测透明带抗体检测试剂的可能。     

自然流产妇女的丈夫精子染色体分析

应用人精子与去透明带金黄地鼠卵受精技术制备人精子染色体标本。对１０例有自然流产史妇女的丈夫共２３０个精子核型进行了分析,并与正常可育男性的２１０个核型对照。结果显示,流产组的精子染色体数目异常率为３９％,与正常组相比（２８％）,两者无显著性差异（Ｐ＞０．０５）;但流产组中结构异常的断裂和无着丝粒断片的比率（６０％;９５％）显著性高于对照组（２５％;２８％）。     

Identification of the mineralocorticoid receptor in human spermatozoa

Aldosterone seems to play a role in the regulation of the electrolyte content of sperm and in the motility of spermatozoa. The aim of the study was to evaluate the presence of the mineralocorticoid receptor (MR) in human ejaculated spermatozoa. We have assayed MR on spermatozoa of freshly ejaculated sperm from healthy donors. The identification of MR was made by using immunohistochemistry and immunofluorescence analyses, while MR mRNA expression was evaluated by real-time PCR assay. The immunohistochemical and immunofluorescence analyses showed positive staining both in the midpiece and in the tail of the spermatozoa. Relative quantification of MR by using real-time PCR shows that the mRNA expression of MR in spermatozoa is lower than in mononuclear leukocytes (positive controls). Sequencing showed complete identity between the sequence obtained from spermatozoa and the human MR cDNA sequence. Further studies should be performed in order to elucidate a possible physiological role of aldosterone in regulating electrolyte concentration, and the pro-oxidant effect of excess aldosterone in this new target tissue.     

Human sperm non-nuclear progesterone receptor expression is a novel marker for fertilization outcome

In a prospective, blind study, we have examined the relationship among the expression of human sperm surface progesterone receptors, the ability to undergo a mannose-stimulated acrosome reaction and the rate of fertilization in vitro. Individual aliquots of motile spermatozoa were surface-labelled with progesterone and/or mannose-fluoresceinated ligands. Spontaneous acrosome loss and the increase in acrosome reactions following exposure of spermatozoa to mannose ligands were assessed using rhodaminated Pisum sativum agglutinin. Progesterone fluoresceinated ligand binding was observed to occur in two patterns: (i) a uniform distribution of labelling over the acrosome cap (pattern II), and (ii) labelling limited to the equatorial and postacrosomal regions of the human sperm head (pattern III). A conversion of pattern II to pattern III binding was observed and was associated with the acrosome reaction. Pattern III binding was highly correlated with both fertilization potential and the ability to undergo a mannose-stimulated acrosome reaction (P < 0.001). In contrast, normal sperm mannose receptor expression was seen in five men whose abnormal progesterone receptor expression/function and inability to acrosome react after mannose treatment were correlated with their reduced fertility in vitro. In conclusion, surface progesterone receptor aggregation enhances the mannose ligand-stimulated acrosome reaction. Such detection of defective sperm surface progesterone receptor expression/function may be useful in the evaluation and management of male infertility.      

CD4 independent binding of HIV gp120 to mannose receptor on human spermatozoa

OBJECTIVE: To characterize the CD4-independent HIV-binding protein of 160 kDa on human spermatozoa. METHODS: The N-terminal amino acid sequence of the 160 kDa protein and its peptide obtained by tryptic digestion were determined. Polymerase chain reaction amplification of human testicular cDNA was performed using degenerate primers corresponding to peptide sequences of the 160 kDa protein. Localization of 160 kDa protein on sperm was performed using fluorescently labeled gp120, followed by inhibition experiments using antagonists to determine the specificity. RESULTS: The partial cDNA sequence of the 160 kDa protein demonstrated 99% identity with human macrophage mannose receptor. Sequence of testicular mannose receptor was obtained and exhibited 99% identity with that of macrophage mannose receptor. Furthermore, mannose receptor protein from sperm extract was found to have a molecular weight of 160 kDa, congruent with that of 160 kDa HIV-binding protein. gp120 binding and mannose receptor expression were localized to the equatorial segment in 10% of ejaculated sperm, which increased after capacitation. Mannan at molar excess concentrations completely inhibited gp120 binding to sperm. CONCLUSIONS: The 160 kDa, CD4-independent HIV-binding sperm protein has been identified as the human mannose receptor protein. The role of mannose receptor in HIV transmission and association with risk of sexual transmission merit further investigation.     

The presence of functional mannose receptor on macrophages at the maternal-fetal interface

BACKGROUND: The mannose receptor (MR) is involved in the initiation of the immune response and regulation of homeostasis during inflammation and tissue remodeling. METHODS: Distribution, endocytosis and possible natural ligand tumor associated glycoprotein-72 (TAG-72) for the MR have been examined by immunohistology, immunocytochemistry and flow cytometry at the maternal-fetal interface, characterized by extensive tissue remodeling. RESULTS: Contrary to disseminated distribution of the MR positive (MR+) cells in term placenta, the MR+ cells of early pregnancy decidua intimately surrounded glands and followed tissue distribution of CD14 positive cells. The mannose receptor was present on freshly isolated first trimester decidual mononuclear cells and distributed mostly on macrophages (77.08 +/- 10.55%, mean +/- SD). The expression of the MR on CD14 positive cells decreased following 18 h culture (P < 0.01) and was accompanied by the reduction of fluorescein isothiocyanate (FITC)-dextran uptake. PAM-1 anti-MR antibody, mannan and TAG-72 reduced FITC-dextran uptake by decidual macrophages. CONCLUSIONS: These data indicate that the MR+ macrophages, surrounding early decidual glands, are able to internalize ligands for carbohydrate recognition domain of the receptor, including decidual secretory phase mucin TAG-72.     

The role of mannose receptor on HIV-1 entry into human spermatozoa

In this opinion article we consider the possibility that human spermatozoa have receptors for human immunodeficiency virus-1 (HIV-1). It is clear that sperm cells have the potential for transmitting HIV-1, but the mechanisms responsible for spreading or the virus by this vector are not known. In contrast to the traditional HIV-1 target cells, spermatozoa do not express CD4 receptors or the CCR5/CXCR4 co-receptors. Recent evidence indicates that astrocytes, which also do not express these molecules, can be infected with HIV-1 through the mannose receptor. Furthermore, a 160-kDa sperm receptor that interacts with the HIV gp 120 has been described. Therefore, we hypothesize that the mannose receptor, of 165-175 kDa, is the receptor that HIV-1 uses to invade spermatozoa, which could lead to both vertical and horizontal transmission of HIV-1.     

Analysis of mannose receptor regulation by IL-4, IL-10, and proteolytic processing using novel monoclonal antibodies

The study of the murine macrophage mannose receptor (MR) has been hampered by the lack of specific reagents. We have generated and characterized novel anti-MR monoclonal antibodies and used them to analyze MR expression in primary mouse macrophages (M(phi)). In BioGel- and thioglycollate-elicited M(phi), interleukin (IL)-4 up-regulated total cell-associated MR (cMR), correlating with enhanced surface expression. We investigated the influence of IL-10, a well-characterized deactivator of M(phi) function, on MR levels and observed that it had a similar effect to IL-4. In both cases, enhanced cMR levels translated into increased production of the soluble form of the receptor (sMR). We have demonstrated the presence of sMR in cultures of stable non-M(phi) transductants expressing full-length MR, indicating that the proteolytic activity responsible for cMR cleavage is not M(phi)-restricted. These data support a role for the MR in T helper cell type 2 cytokine-driven, immune responses and suggest a non-M(phi) contribution to sMR production in vivo.     

Co-Expression of Mannose-Ligand and Non-Nuclear Progesterone Receptors on Motile Human Sperm Identifies an Acrosome-Reaction Inducible Subpopulation

PROBLEM : To determine whether surface expression of receptors for progesterone and mannose can be used to identify spermatozoa likely to undergo an acrosome reaction after zona binding and to compare the reactivity of these receptors with naturally occurring sperm head-directed anti-sperm antibodies (ASAs). METHOD : Progesterone binding sites on the surface of fresh and capacitated motile human sperm in relation to acrosome status were visualized using a cell-impermeant progesterone. Free progesterone and/or mannose ligands were compared for percent sperm binding and ability to induce an acrosome reaction. Western blots of sperm proteins localized to the plasma membrane and surface proteins precipitated following passive transfer of serum ASAs were probed with progesterone-horseradish peroxidase. The effects of the same ASAs on ligand binding and on the induced acrosome reaction were examined. RESULTS : The two receptors are located in close proximity on a subset of capacitated motile sperm and are coordinately cleared from the plasma membrane overlying the acrosomal cap prior to exocytosis. The surface appearance of functional binding sites for each ligand, however, is regulated by different mechanisms and the progesterone receptor alone is specifically precipitated by ASAs. Passive transfer of ASAs to capacitated sperm selectively inhibits the progesterone-stimulated acrosome reaction but not the ionomycin-induced acrosome reaction or the ability of sperm to bind mannose ligands. CONCLUSIONS : Sperm from fertile donors incubated under capacitating conditions in vitro can be subdivided into acrosome reaction inducible and noninducible subpopulations on the basis of the co-expression or total absence of these receptors. The combined data indicate that reaction of sperm surface progesterone receptors with ASAs contributes to the acrosome reaction insufficiency observed in anti-sperm immune infertility.     

Characterization of functional mannose receptor in a continuous hybridoma cell line

ABSTRACT: BACKGROUND: The mannose receptor is the best described member of the type I transmembrane C-type lectins; however much remains unanswered about the biology of the receptor. One difficulty has been the inability to consistently express high levels of a functional full length mannose receptor cDNA in mammalian cells. Another difficulty has been the lack of a human macrophage cell line expressing a fully functional receptor. Commonly used human macrophage cell lines such as U937, THP-1, Mono-Mac and HL60 do not express the mannose receptor. We have developed a macrophage hybridoma cell line (43MR cells) created by fusion of U937 cells with primary human monocyte-derived macrophages, resulting in a non-adherent cell line expressing several properties of primary macrophages. The purpose of this study was to identify and select mannose receptor-expressing cells using fluorescence-activated cell sorting and to characterize the expression and function of the receptor. RESULTS: In the current study we show that the mannose receptor found on this novel cell has endocytic characteristics consistent with and similar to the mannose receptor found on the surface of monocyte-derived human macrophages and rat bone marrow-derived macrophages. In addition, we demonstrate that these cells engage and internalize pathogen particles such as S. aureus and C. albicans. We further establish the transfectability of these cells via the introduction of a plasmid expressing influenza A hemagglutinin. CONCLUSIONS: The 43MR cell line represents the first naturally expressed MR-positive cell line derived from a human macrophage background. This cell line provides an important cell model for other researchers for the study of human MR biology and host-pathogen interactions.     

Use of mannose ligands in IVF screens to mimic zona pellucida-induced acrosome reactions and predict fertilization success

A predictive test for determining whether motile populations of human spermatozoa will fertilize eggs in vitro has been an elusive goal of clinical research. We have developed an assay for the ability of motile human spermatozoa to bind fluorescein isothiocyanate-labelled mannosylated bovine serum albumin (Man-FITC-BSA) as a test for the presence of sperm surface receptors (lectins) for mannose ligands. Mannosylated ligands are present on the human zona pellucida and are involved in the species-specific binding of human spermatozoa to the zona pellucida. We now demonstrate in prospective blinded analysis that the fractional increase in acrosome loss following a mannose ligand challenge is highly correlated with the rate of fertilization in vitro. Using an incremental increase of acrosome exocytosis of >0.1 as a threshold to predict which specimens will yield normal fertilization, the assay has a sensitivity of 97.8%, a specificity of 83.3%, a positive predictive value of 95.7% and a negative predictive value of 90.7%. These data indicate that testing for a mannose-induced acrosome reaction may be useful in assessment of sperm function prior to in- vitro fertilization in order to assign males to conventional insemination or intracytoplasmic sperm injection protocols.      

考马斯亮蓝染色法检测人精子顶体反应的评价

为建立一种检测人精子顶体反应的简便实用的新技术，用３．５％高氯酸水溶液配制０．０５％考马斯亮蓝（Ｒ２５０）染色液浸染人精子３０ｍｉｎ，顶体完整者顶体区染成紫蓝色，顶体反应者则不染。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。方法学检验证明新技术结果可靠。本法操作简便、经济，普通显微镜即可检测，易于推广，克服了原有技术复杂昂贵的缺点，具有研究和临床诊断的良好价值。