鲫鲤杂种F2精子多态性的研究

本文利用扫描电镜和透射电镜对鲫鲤杂种F2精子的超微结构进行了系统的研究。F2精子由头部、中片和尾部组成,头部前端有液泡,没有顶体。研究发现,F2精子头部大小差别明显,最小的精子头径只有1．32μm,而最大的那个“超级精子”头径约18．39μm,但是绝大部分精子头径在1．85-2．15μm。另外,通过透射电镜还发现了双核精子和双尾精子。结果表明,F2精子中存在着明显的多态性,其中有正常的精子,即单倍体精子;也有异常的精子,包括二倍体精子、四倍体精子甚至更高倍性的精子,还有非整倍体精子。本研究结果为二倍体的精子和二倍体的卵子结合产生四倍体的机制提供了有利的证据。     

鲫鲤杂种F2精子多态性的研究

本文利用扫描电镜和透射电镜对鲫鲤杂种F2精子的超微结构进行了系统的研究.F2精子由头部、中片和尾部组成,头部前端有液泡,没有顶体.研究发现,F2精子头部大小差别明显,最小的精子头径只有1.32μm,而最大的那个"超级精子"头径约18.09μm,但是绝大部分精子头径在1.85-2.15μm.另外,通过透射电镜还发现了双核精子和双尾精子.结果表明,F2精子中存在着明显的多态性,其中有正常的精子,即单倍体精子;也有异常的精子,包括二倍体精子、四倍体精子甚至更高倍性的精子,还有非整倍体精子.本研究结果为二倍体的精子和二倍体的卵子结合产生四倍体的机制提供了有利的证据.     

与顶体反应相关的抗人精子单克隆抗体及其抗原的特性 Ⅰ顶体反应的诱导及抗顶体反应精子的单克隆抗体的制备

精子必须经过获能和顶体反应(AR)才具受精能力。利用获能和AR 前后精子细胞内和精子表面大分子的变化,可探索新的避孕办法和男性不育诊断及治疗的新途径。本文将正常人精子于体外在BWW-BSA 培养基中获能,用钙离子载体A 23187诱导人精子进行AR(以三重染色和金霉素荧光染色两种方法检测这些精子的AR 率为50%左右),然后用这些新鲜的人精子作为免疫原,制备了23个抗人精子单克隆抗体,其中21个为IgM,2个分别为IgG_1和IgG_2。根据23个单克隆抗体与经获能和AR 处理的精子以及未处理精子的不同免疫反应,将它们分为A(AR 精子反应组)、B(未处理精子反应组)和C(双重精子反应组)三组,并测定了这些单克隆抗体与人的某些白血病细胞系的交叉反应。     

生精细胞及精子赖氨酸含量的研究

本实验取１０只Ｗｉｓｔａｒ大鼠的睾丸和附睾,睾丸石蜡切片,附睾精子涂片后用苯胺蓝染色显示赖氨酸含量。结果是睾丸生精小管中精原细胞和精母细胞染色较深即赖氨酸含量较高,精子细胞和精子染色渐淡即赖氨酸含量降低,而附睾精子显示,在附睾头部的精子染色较深,附睾尾部的精子几乎不着色,应用显微分光光度计测定附睾精子,计算出头部的精子赖氨酸含量在１左右,尾部的精子赖氨酸含量接近于零。本实验还检测了１０例正常人及１０例不育者精子的赖氨酸,结果为正常人精子的赖氨酸含量较低,不育者精子赖氨酸含量高且畸形率也高。提示精子赖氨酸含量高是核蛋白转型异常的征象,可能是男性不育的一个重要原因。     

精子尾部发育相关蛋白研究进展

精子尾部结构与其运动功能密切相关。精子的运动能力直接决定了精子能否正常运输到输卵管与卵子受精。精子尾部的形成和发育是一个极其复杂的过程,由多种蛋白质精细调控。研究发现,多种精子尾部发育相关蛋白的缺陷可导致少、弱、畸形精子症。本文根据精子尾部的超微结构顺序,对近年来精子尾部发育相关蛋白的研究进展进行综述,以期为男性不育症的遗传学诊断和治疗提供理论基础和实践的可能。     

精子的运动特性

精子的运动特性与生育力有密切关系。本文从超微结构水平和分子生物学基础出发,阐述精子运动机制的微管滑动学说;介绍了精子在液体介质中的运动形式、检测精子活动力的主要技术、影响精子活动力的因素、从异常精液中选择正常精子的技术,以及精子活动力在雄性和雌性生殖道中的演变过程。     

SRC激酶和磷酸酶PP1γ2/PP2A的相互作用对小鼠附睾精子成熟及运动的调控

目的哺乳动物附睾精子成熟、运动能力的获得与维持是保证精子执行正常功能、完成受精的前提和基础,但调控此过程的机制仍未完全阐明。SRC激酶参与小鼠精子获能的调控,Ser/Thr磷酸酶PP1γ2/PP2A是调控小鼠精子成熟、运动性获得的关键酶,但二者是否具有相互作用且这种相互作用是否调控着精子运动并不清楚。为此,本研究探究了小鼠精子中SRC激酶与磷酸酶PP1γ2/PP2A的关联性及其对精子运动的调控作用,旨在阐明其调控精子运动的作用机制。方法通过Western Blot技术、酶活性分析技术以及免疫共沉淀技术分析昆明小鼠附睾头和附睾尾精子苏氨酸磷酸化水平、SRC激酶活性、附睾头和附睾尾精子酶活性和SRC激酶与Ser/Thr磷酸酶相互关系,探讨SRC激酶抑制剂(SU6656)和激活剂(sc-3052)分别对附睾尾、附睾头精子磷酸酶活性和运动度的影响。结果附睾尾精子苏氨酸磷酸化水平高于附睾头精子;附睾头精子中的SRC激酶活性低于小鼠附睾尾精子;附睾头精子磷酸酶活性显著高于附睾尾精子磷酸酶活性(P0.05);附睾尾精子中SRC激酶与PP1γ2/PP2A具有关联性作用,进而调控精子活力;在SRC激酶活性较高的附睾尾精子中,添加SU6656,磷酸酶PP1γ2/PP2A活性随之增加,精子活力下降;在SRC激酶活性较低的附睾头精子中,添加sc-3052,磷酸酶PP1γ2/PP2A活性随之下降,而精子活力增加。结论小鼠附睾头与附睾尾精子中SRC激酶和磷酸酶PP1γ2/PP2A的活性具有显著差异;小鼠精子中SRC激酶与PP1γ2/PP2A具有相互作用,呈负相关性,即SRC激酶通过抑制精子中磷酸酶PP1γ2/PP2A活性以调控精子运动。     

用原子力显微镜观察人类精子的表面结构

目的:探讨用原子力显微镜观察精子表面结构的方法。方法:经常规洗涤正常人精液后进一步除去精子表面和生理溶液中的蛋白质,直接用原子力显微镜观察人类精子的表面形态。结果:获得了人类精子表面的细微结构和精子头的三维数据。精子全长约47μm,精子头约4.6μm,顶体位于精子头前端1/2~2/3,顶体前端扁平。精子赤道区有两圈环形凸起。结论:不需特殊处理,用原子力显微镜能直接观察精子表面的超微结构,并获得量化的三维数据。     

精子端粒长度与特发性男性不育相关

端粒是真核生物染色体末端的多功能特异性DNA-蛋白结构,覆盖在染色体末端,保护基因组的稳定性。端粒在减数分裂过程中起到了十分重要的作用,协助染色体配对、联会、同源重组和分离。精子中的端粒可能在精子的受精能力和胚胎发育中起到重要作用。近年来,端粒与生殖的相关性研究成为一个新的热点,但精子端粒与男性不育间的相关性并不明确。本文采用实时荧光定量PCR方法检测中国特发性男性不育人群(126例)和正常可育男性人群(138例)的精子相对端粒长度,结果发现,特发性男性不育病例的精子平均相对端粒长度(2.894±0.115)低于正常对照组(4.016±0.603),差异具有统计学意义(P=5.097×10^-5);并且精子相对端粒长度与精子密度、精子总数和精子活力都有显著的相关性：精子数量较多和/或精子活力较高,精子相对端粒长度较长。研究结果提示,在中国人群中,精子端粒长度与特发性男性不育具有相关性,精子的端粒长度可能影响精子发生和精子的功能,精子端粒的缩短导致精子数目及活力的降低从而导致男性不育。     

中国蟾蜍属精子形态比较

采用扫描电镜和光学显微镜观察了采自我国的蟾蜍属(Bufo)7种(亚种)的精子形态,对精子各部位量度进行了测量和计算。结果表明,该属7种(亚种)精子的形态基本相同,精子由头部、中片和尾部组成,头部细长微弯且前端渐尖,中片有球状突起,尾部长,由轴纤维、轴丝和波动膜构成。与已有报道的两栖动物的精子形态相比较,蟾蜍属精子与无尾类其他科精子形态差别较大,而与有尾类精子形态相似。本文认为两栖动物精子形态和量度在科间存在明显差异;两栖动物精子形态的差异可能与其繁殖模式有关。     

A laser scanning confocal microscopy method. Simultaneous detection of intracellular Ca2+ and apoptosis using Fluo-3 and Hoechst 33342

OBJECTIVE: To develop a simple and direct method to simultaneously determine apoptotic cells from a treated population of cells and detect the changes of intracellular Ca2+ in these apoptotic cells, in particular single ones, by confocal microscopy. STUDY DESIGN: MGC-803 cells treated with As2O3 were used as the double-staining cell model with Hoechst 33342 as a DNA probe and Fluo-3AM as a Ca2+ indicator. MGC-803 cell apoptosis induced by As2O3 was first demonstrated by DNA ladder in gel electrophoresis. Based on the difference in DNA stainability with Hoechst 33342 and corresponding fluorescence intensity between live and apoptotic cells, apoptotic cells and the changes in intracellular Ca2+ were detected at the same time by confocal microscopy. No necrotic cells in the group treated with As2O3 were found by the trypan blue exclusion test. RESULTS: The results from confocal microscope detection showed that intact and apoptotic cells were successfully recognized and the changes of intracellular Ca2+ in apoptotic and intact cells were simultaneously detected in the same sample. CONCLUSION: We provided a useful method to exactly detect changes in intracellular Ca2+ in apoptotic cells, especially in single ones, by confocal microscopy and to exclude the artifact effect of necrotic and intact cells.     

Immunolocalization of laminin during estrogen-induced differentiation of quail oviduct epithelial cells

During estrogen-induced development of the quail oviduct, tubular glands are formed by evagination of epithelial cells into the stroma. The distribution of laminin was studied during the early stages by means of immunofluorescence and immunoperoxidase techniques. Ultrastructural changes in the basal lamina were studied by electron microscopy. Basement membranes at all stages of development were delineated with 3 polyclonal antilaminin antisera. However, in ovariectomized birds, laminin could not be detected by one of the polyclonal antilaminin antisera. Subsequently, this antibody detected laminin as epithelial cell evaginations were induced by estradiol benzoate. The heavy and light chains of Engelbreth Holm sarcoma (EHS) laminin were revealed in immunoblotting by all antibodies. By electron microscopy after the immunoperoxidase technique with antilaminin antisera laminin appears to be accumulated mainly in the lamina densa. Furthermore, the thickness of the basal lamina increases during oviduct development. These data indicate that basal lamina organization is modified during oviduct cell differentiation and that immunoreactivity of epithelial basement membrane laminin changes during development.     

Synthesis of eicosanoids by gamma-interferon-differentiated U937 cells

Interferons induce morphological, biochemical and functional alterations in monocyte macrophage and myeloid cell lines. We studied the effect of 3 days incubation with gamma-interferon from human buffy coats on the global synthesis of arachidonic acid metabolites by U937 cells. Interferon-induced morphologic changes including cytoplasmic and nuclear changes and the appearance of multiple lysosomal-like granules consistent with cellular differentiation were observed by electron microscopy. The labeling of phosphatidylserine, phosphatidylcholine and phosphatidylethanolamine was increased and that of phosphatidylinositol, free fatty acids as 3H-arachidonic acid and neutral lipids reduced, when interferon-treated cells were incubated with 3H-arachidonic acid. Interferon caused qualitative and quantitative changes in the synthesis of cyclooxygenase and lipoxygenase products. A23187, a calcium ionophore, and the tumor promotor, phorbol myristate acetate, greatly increased the synthesis by interferon-differentiated cells of 2 cyclooxygenase products; synthesis of lipoxygenase products was reduced. In the presence of indomethacin, 'shunting' into putative lipoxygenase products occurred. The relationship between interferon-induced morphologic and functional changes, the development of altered phospholipid and eicosanoid metabolism and the identity of these metabolites are yet to be established.     

红豆草根瘤侵染细胞的超微结构变化

红豆草(Onobrychis viciaefolia Scop.)是一种抗旱、耐寒、耐热和耐瘠薄的优良豆科牧草,不仅是很好的饲料和绿肥,而且还能大量结瘤固氮,提高土地肥力。因此,它在我国甘肃和其他北方干旱和半干旱贫瘠地区广为种植。虽然不少学者曾对它的引种条件、生产性能、营养成分和形态解剖作过许多研究,但对其共生固氮,特别是与共生固氮息息相关的根瘤在发育中的变化却至今尚无系统报道。因此,为了更好开发利用这一资源,     

Ontogenetic changes in location and morphology of chloride cells during early life stages of the Nile tilapia Oreochromis niloticus adapted to fresh and brackish water

Ontogenetic changes in the location, size, density and morphology of chloride cells in the Nile tilapia Oreochromis niloticus adapted to fresh and brackish water are described using Na(+) /K(+) -ATPase immunohistochemistry, light microscopy (LM), scanning electron microscopy (SEM) and confocal scanning laser microscopy (CSLM). The pattern of chloride cell distribution changed during development under both treatments, with chloride cell density decreasing significantly from hatch to 7 days post-hatch, but appearing on the inner opercular area at 3 days post-hatch and increasing significantly thereafter (P < 0·05). Chloride cells were always denser in fresh- than in brackish-water larvae. In both treatments, chloride cells located on the outer operculum and tail showed a marked increase in size with age, but cells located on the abdominal epithelium of the yolk sac and the inner operculum showed a significant decrease in size (P < 0·05). Chloride cells from brackish-water adapted larvae from 1 day post-hatch onwards were always significantly larger (P < 0·05) than those from freshwater-adapted larvae. SEM revealed structural differences in chloride cell apical morphology according to environmental conditions. There appears to be clearly defined temporal staging of the appearance of adaptive mechanisms that confer an ability to cope with varying environmental conditions during early development.     

Immuno-gold localization of IAA in leaf cells of Prunus persica at different stages of development

Subcellular localization of indole-3-acetic acid (IAA) in leaf cells of peach ( Prunus persica [L.] Batsch 'Hakuho') was investigated using imuno-gold electron microscopy. The distribution pattern of the gold particles, which detected IAA, changed as cells matured. The most prominent feature was the accumulation of the gold label in the chloroplasts and mitochondria of the parenchyma cells of opened leaves. Throughout the development, the cytosol, nuclei, and cell wall were labelled, although the level was low and no significant changes occurred. The density of colloidal gold at each stage of leaf development was well correlated with the analytical data obtained by high-performance liquid chromatography (HPLC).     

Morphological and functional changes in Lewis lung carcinoma cells after treatment with A23187 calcium ionophore

Cultured Lewis Lung carcinoma cells (3LL) treated with A23187 calcium ionophore were studied by transmission electron microscopy and HPLC analysis. Results showed that A23187 calcium ionophore induces on osmiophilic inclusion bodies of 3LL cells similar process of lamellar bodies formation to those reported in the development of the osmiophilic inclusions bodies within the granular pneumocyte of normal lungs. HPLC analysis shows that calcium ionophore generates quantitative changes in the 3LL cytoplasmic protein content expressed by an increase of 18-22 kDa and 4-9 kDa proteins.     

Influence of membrane active substances on cell surface morphology of cultured aortic endothelial cells

1. The changes occurring on the surface of cultured aortic endothelial cells under the influence of exogenous cholesterol, lysolecithin, or cholesterol + lysolecithin were pursued by scanning and transmission electron microscopy. 2. Both compounds were found to cause surface changes under different quantitative relations, as shown by scanning electron microscopy of unfixed preparations. The changes were less significant in preparations fixed prior to the scanning--or transmission electron microscopic examination. 3. Other cell lines maintained in this laboratory and similarly treated with cholesterol, were found to be less responsive than the endothelial cells. 4. Cholesterol content was determined by thin layer chromatography in six different cell lines, before and after the addition of cholesterol. Only the endothelial cells showed a notable rise of cholesterol content after treatment. This fact may confirm our finding that cholesterol induced morphological changes were demonstrable only in the endothelial cells.     

Effects of lemongrass oil on the morphological characteristics and peptidoglycan synthesis of Escherichia coli cells

The antibacterial effect of lemongrass oil, obtained from the aerial part of Cymbopogon citratus, on cells of Escherichia coli was investigated by electron microscopy and by measuring cell wall formation. Two strains of E. coli K-12 were used, one of which required diaminopimelic acid in the growth medium for its murein formation. Lemongrass oil was found to elicit morphological changes like filamentation, inhibition of septum formation, spheroplast formation, production of 'blisters', 'bulges' or mesosomes, as well as lysis and development of abnormally shaped cells. The incorporation of radioactively labelled diaminopimelic acid into the cell wall murein of strain W7, was inhibited by lemongrass oil in a dose dependent way. The sequence of changes induced by lemongrass oil on bacterial cell morphology and also its interference with murein synthesis in E. coli cells were interpreted to involve the penicillin binding proteins PBP 2 and PBP 3.     

Morphological changes induced by the calcium ionophore A23187 in rat basophilic leukemia (2H3) cells.

RBL-2H3 cells have been widely used to study histamine release in vitro. It was previously shown that these cells undergo striking morphological changes after IgE-mediated secretion. The present study was undertaken to examine if the morphological changes were dependent on activation of the Fc epsilon receptor. Therefore, the cells were stimulated to release histamine by two different mechanisms: activation of the Fc epsilon receptor by antigen and treatment with the calcium ionophore A23187. Cell surface and cytoskeletal changes were examined by fluorescence microscopy and scanning electron microscopy after either IgE- or ionophore-mediated histamine release. After exposure of the cells to either secretagogue, the cells spread over the surface of the culture dish and underwent rearrangement of the cytoskeleton. In addition, scanning electron microscopy revealed that deep ruffles developed on the surface of the cells undergoing IgE-mediated release. The surface changes were not as pronounced with the ionophore. The distribution of the cytoskeletal elements was examined by immunofluorescence using FITC-phalloidin and antibodies against vimentin and tubulin. In unstimulated cells actin was localized at the cell periphery, just under the plasma membrane. In the stimulated cells it was associated with the cell periphery and concentrated in the surface ruffles. As the stimulated cells spread, intermediate filaments and microtubules became distributed throughout the cell body, but there was no obvious association with the membrane ruffles. These morphological changes were dependent on the presence of extracellular calcium and on the concentration of ionophore or antigen, and were also correlated with the amount of histamine released. Additionally, IgE-mediated stimulation led to increased uptake of the soluble-phase tracer Lucifer yellow, whereas stimulation with the ionophore A23187 showed no increase in Lucifer yellow internalization. Ionophore A23187 produced changes similar but not identical to those seen in the RBL-2H3 cells after IgE-mediated histamine release. The differences may be owing to the involvement of the Fc epsilon receptor in IgE-mediated secretion.