Plasma membrane structure in spermatogenic cells of the swordtail (teleostei,Xiphophorus helleri)

The plasma membrane of spermatogenic cells of the teleost Xiphophorus helleri was examined ultrastructurally and cytochemically in order to characterize the temporal development of the membrane specializations characteristic of the mature spermatozoon. Mature sperm display a mosaic distribution of Concanavalin A and Ricinus comrnunis I binding sites; the anterior region of the head displays an intense binding that is not seen in other surface regions. This asymmetric binding is evident in early spermatids and the area of lectin binding appears associated with the plasma membrane overlying the nucleus. Transmission electron microscopy reveals that the plasma membrane over the anterior region of the head is characterized by an ordered glycocalyx and a tight adherence to the underlying nucleus. Additional membrane differentiations were revealed both in the midpiece region where a “submitochondrial net” is attached to the plasma membrane and at the base of the axoneme where the plasma membrane possesses a “collar-like” arrangement of circumferential rings. The possible functions of these differentiations, as well as their correlation to differentiations seen in sperm of other animal groups, are discussed.     

Structural effects of titanium citrate on the human erythrocyte membrane

The structural effects of titanium citrate on the human erythrocyte membrane were studied through its interaction with intact erythrocytes and isolated unsealed human erythrocyte membranes (IUM). The studies were carried out by scanning electron microscopy and fluorescence spectroscopy, respectively. Titanium citrate induced shape changes in erythrocytes, which were damaged and ruptured leaving empty and retracted membranes. Fluorescence spectroscopy measurements in IUM indicated a disordering effect at both the polar head group and the acyl chain packing arrangements of the membrane phospholipid bilayer. Titanium citrate also interacted with molecular models of the erythrocyte membrane consisting in bilayers of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE), representing classes of phospholipids located in the outer and inner monolayers of the erythrocyte membrane, respectively. X-ray diffraction indicated that titanium citrate induced structural perturbation of the polar head group and of the hydrophobic acyl regions of DMPC, while the effects on DMPE bilayers were negligible. This conclusion is supported by fluorescence spectroscopy measurements on DMPC large unilamellar vesicles. All these findings indicate that the structural perturbations induced by titanium to human erythrocytes can be extended to other cells, thereby affecting their functions.     

Membrane specializations in the paired spermatozoa of dytiscid water beetles

The paired spermatozoa of the dytiscid beetles Dytiscus marginalis and Hydaticus seminiger were studied by electron microscopy with the aim of examining whether the regions of the cell membrane in the zones of sperm conjugation might differ from other regions and to explore whether these cells had any other specialized domains of the cell membrane that could be recognized by the freeze-fracturing technique. The spermatozoa are conjugated along one side of the sperm head and proximal tail portion, called the ventral side. The cell membrane was seen to contain tightly packed intramembranous particles (IMPs) that were predominantly located in the external membrane face (the E-face). In thin sections the cell membrane had a ladder-like appearance at these regions and a specialized type of glycocalyx seen as a fluffy material containing granules. Other specialized membrane domains could also be recorded: a ribbon of particles in the protoplasmic face (P-face) of the dorsal side of the spermatozoon at the proximal tail portion and regularly arranged particle rows in the P-face of the distal tail portion. These domains corresponded to regions where the glycocalyx is prominent. Both the E-face and the P-face of the cell membrane were seen to contain numerous intramembranous particles, which suggests an active function for both membrane leaflets; this is in contrast to the situation in most cells where the particles are mainly in the P-face. The functions of the intramembranous particles in the specialized domains of the cell membrane remains unknown. Some particles may represent receptors or ion gates, others proteins with a mechanical function.     

Developmental Ultrastructure of Glandular Trichomes of <Emphasis Type="Italic">Rosmarinus officinalis</Emphasis>: Secretory Cavity and Secretory Vesicle Formation

Glandular trichomes in the leaf lamina of Rosmarinus officinalis L. were examined by scanning and transmission electron microscopy. The leaves were characterized by an abundance of two types of glandular trichomes—small capitate and large peltate glandular trichomes. In addition to the glandular trichomes, numerous non-glandular trichomes were present on the abaxial surface of the leaf. These trichomes mainly predominated on the midrib, whereas glandular trichomes occurred on non-vein areas. At the initial phase of secretory cavity formation, hyaline areas were abundant in periclinal walls of head cells, while they were not observed in the anticlinal walls. The hyaline areas gradually increased in size, fusing with other areas throughout the wall. Loose wall material adjacent to hyaline areas was released from the head cell walls and migrated into the secretory cavities. As the secretory cavities continued to enlarge, the new vesicles emerging into the secretory cavities from the walls of head cells became surrounded with the surface of a typical membrane. They developed a round shape, but the contours of the vesicle surfaces appeared polygonal when tightly packed inside a cavity. These vesicles varied in size; small vesicles often possessed electron-dense contents, while large vesicles contained electron-light contents.     

Membrane movements and fluidity during rotational motility of a termite flagellate. A freeze-fracture study

Freeze-fracture electron microscopy was used to examine the structure of a region of plasma membrane that undergoes continual, unidirectional shear. Membrane shear arises from the continual clockwise rotation of one part (head) of a termite flagellate relative to the rest of the cell. Freeze-fracture replicas show that the lipid bilayer is continuous across the shear zone. Thus, the relative movements of adjacent membrane regions are visible evidence of membrane fluidity. The distribution and density of intramembrane particles within the membrane of the shear zone is not different from that in other regions of the cell membrane. Also, an additional membrane shear zone arises when body membrane becomes closely applied to the rotating axostyle as cells change shape in vitro. This suggests that the entire membrane is potentially as fluid as the membrane between head and body but that this fluidity is only expressed at certain locations for geometrical and/or mechanical reasons. Membrane movements may be explained solely by cell shape and proximity to rotating structures, although specific membrane-cytoskeletal connections cannot be ruled out. The membrane of this cell may thus be viewed as a fluid which adheres to the underlying cytoplasm/cytoskeleton and passively follows its movements.     

Biophysical characterization of genistein–membrane interaction and its correlation with biological effect on cells — The case of EYPC liposomes and human erythrocyte membranes

With application of EPR and ¹H NMR techniques genistein interaction with liposomes formed with egg yolk lecithin and with erythrocyte membranes was assessed. The present study addressed the problem of genistein localization and its effects on lipid membrane fluidity and protein conformation. The range of microscopic techniques was employed to study genistein effects on HeLa cells and human erythrocytes. Moreover, DPPH bioassay, superoxide anion radical test and enzymatic measurements were performed in HeLa cells subjected to genistein. The gathered results from both EPR and NMR techniques indicated strong ordering effect of genistein on the motional freedom of lipids in the head group region and the adjacent hydrophobic zone in liposomal as well as in red blood cell membranes. EPR study of human ghost showed also the changes in the erythrocyte membrane protein conformation. The membrane effects of genistein were correlated with the changes in internal membranes arrangement of HeLa cells as it was noticed using transmission electron microscopic and fluorescent techniques. Scanning electron and light microscopy methods showed that one of the aftermaths of genistein incorporation into membranes was creation of echinocytic form of the red blood cells with reduced diameter. Genistein improved redox status of HeLa cells treated with H₂O₂ by lowering radicals' level.     

日本鳗鲡精卵的超微结构以及受精过程观察

通过扫描电镜和透射电镜对经人工催产获得的日本鳗鲡(Anguilla japonica)精子、卵膜的超微结构以及受精过程进行了观察。实验观察到,除一般硬骨鱼类的精子特性外,日本鳗鲡精子有其独特的结构。精子头部为不规则的梨形,有背腹面之分。一个巨大的球形线粒体位于头部顶端。精子中段向后伸出一支根,支根位于袖套腔外精子的背侧,前端向精子头部线粒体方向延伸,支根的微管结构为"8+2"结构,并在精子入卵过程中起到切断鞭毛的作用。精子的尾部由鞭毛和鞭毛末端的结组成。鞭毛横切面呈圆形,无侧鳍,鞭毛微管结构为"9+0"结构。受精卵的整个表面密布着无规律延伸的脊、脊包围形成的窝和窝中的孔所组成的脊孔复合体,但无典型特征的受精孔。受精卵超薄切片观察发现,日本鳗鲡卵膜分为外层壳膜和内层卵黄膜。壳膜与卵黄膜间为卵周隙。壳膜只观察到放射带,未见透明带。放射带可分为三个亚层:最外层为脊孔复合体的脊,中间层为皱纹层,最内层为致密的平滑层。脊孔复合体的孔横穿整个放射带,在放射带内层形成一个乳突状结构。日本鳗鲡的卵膜不仅具有保护卵子的作用,而且还参与了受精。实验还通过扫描电镜观察了日本鳗鲡精子的入卵过程。观察结果认为:日本鳗鲡精子入卵过程可分为卵膜对精子的吸引、精子对卵膜的锚定、精核的进入和孔封闭等4个阶段。但由于研究只观察到受精过程中日本鳗鲡精子和卵膜的形态变化,因此对精子穿过卵膜的方式和特征等尚需做进一步的研究。整个受精过程为1min30s左右。此外,研究还探讨了日本鳗鲡精子结构的特殊性和受精过程的特殊性,为进一步突破日本鳗鲡人工育苗技术提供了理论依据。       

Morphological detection of filipin-sterol complexes in the cytoplasmic membrane of staphylococcal L-form

Filipin, a sterol-specific antibiotic, and freeze-fracture electron microscopy were used to study the presence and distribution of sterol in the cytoplasmic membrane of stable staphylococcal L-form cells. Fixed cells were treated with filipin, and then observed by freeze-fracture electron microscopy. Freeze-fractured profiles of the L-form cells treated with filipin demonstrated irregular distribution of protuberances or pits of 25-30 nm, representing filipin-sterol complexes, on the proto-plasmic fracture face (PF) and exoplasmic fracture face (EF) of the cytoplasmic membrane. In contrast, no such structure was detected in the filipin-treated parent cells or protoplasts. The results suggest that some sterol molecules, which are usually not found in staphylococcal or other bacterial cells, emerged on the cytoplasmic membrane after the cells were converted to the stable L-form.     

Chloroquine-sensitive transplasmalemma electron transport in Tetrahymena pyriformis: a hypothesis for control of parasite protozoa through transmembrane redox

Plasma membrane electron transport was studied in a protozoan cell, Tetrahymena pyriformis, by assaying transmembrane ferricyanide reduction and the reduction of iron compounds. The rates of ferricyanide reduction varied between 0.5 and 2.5 mumol/g dry wt. per min, with a pH optimum at 7.0-7.5. Other active non-permeable electron acceptors, with redox potentials from +360 to -125 mV, were cytochrome c, hexaammine ruthenium chloride, ferric-EDTA, ammonium ferric citrate, and indigo di-, tri- and tetrasulfonates. It was found that Tetrahymena cells can reduce external electron acceptors with redox potentials at pH 7.0 down to -125 mV. Ferricyanide stimulates ciliary action. Transmembrane ferricyanide reduction by Tetrahymena was not inhibited by such mitochondrial inhibitors as antimycin A, 2-n-heptyl-4-hydroxyquinoline N-oxide, or potassium cyanide, but it responded to inhibitors of glycolysis. Transmembrane ferricyanide reduction by Tetrahymena appears to involve a plasma membrane electron transport chain similar to those of other animal cells. As in other cells, the transmembrane electron transport is associated with proton release which may be involved in internal pH control. The transmembrane redox system differs from that of mammalian cells in a 20-fold greater sensitivity to chloroquine and quinacrine. The Tetrahymena ferricyanide reduction is also inhibited by chlorpromazine and suramin. Sensitivity to these drugs indicates that the transplasma membrane electron transport and associated proton pumping may be a target for drugs used against malaria, Trypanosomes and other protozoa.     

Intrinsic uncoupling of mitochondrial proton pumps. 1. Non-ohmic conductance cannot account for the nonlinear dependence of static head respiration on delta microH

The passive membrane conductance LH1 of rat liver mitochondria has been measured and compared with the quantity nJesh/delta microHsh (n = H+/e stoichiometry; Jesh = rate of electron transfer in static head) over a delta microH range. The two curves approach each other only in the lower part of the range, while they sharply diverge at large values of delta microH. Thus nJesh/delta microHsh cannot be considered to be a measure of LH1 in the upper delta microH region. Only a fraction of the static head electron flow is accounted for by futile proton cycling via leaks. Contaminating open membrane fragments or completely leaky mitochondria can be responsible for only a small part of the residual rate of oxygen consumption. We conclude that a large part of static head respiration must have yet another cause and propose it to be intrinsic uncoupling of the respiratory chain enzymes.     

THE SURFACE EVENTS AT FERTILIZATION OF THE SEA URCHIN EGG I. EVENTS ON THE SURFACE OF THE VITELLINE COAT1

Sperm-egg interaction during normal fertilization in the sea urchins, Strongylocentrotus intermedius and Hemicentrotus pulcherrimus, was studied by scanning and transmission electron microscopy. Several seconds after insemination, acrosome-reacted spermatozoa were found attached to the surface of the vitelline coat on each egg. Soon, several bulges of the vitelline coat appeared surrounding the fertilizing spermatozoon. These bulges then spread over the surface increasing in number, while they became fewer and disappeared around the sperm head. Thin sections of the bulging areas revealed discharging cortical granules. As the bulging vitelline coat was elevated, the sperm head was incorporated into the perivitelline space, passing through a small hole in the coat that resulted from penetration of the sperm acrosomal process immediately before fusion of the gametes. When the spermatozoon disappeared beneath the fertilization membrane, a hole was left in the membrane and the cortical reaction had finished on the other hemispheric surface. Mechanical removal of the membrane at that time exposed a spermatozoon protruding perpendicularly from the egg plasma membrane surface. The anterior tip of the sperm head was smoothly connected with the egg surface, and neither microvillous projections nor cytoplasmic covering of the egg cytoplasm could be found around the spermatozoon.     

20-羟基蜕皮甾酮处理后舞毒蛾外部形态和幼虫体壁超微结构的变化

为了探明20-羟基蜕皮甾酮对昆虫蜕皮过程中体壁的表皮层、 皮细胞及其细胞器的具体影响过程, 本研究利用透射电镜技术研究了20-羟基蜕皮甾酮对舞毒蛾Lymantria dispar （Linnaeus）5龄幼虫体壁超微结构的变化。结果表明, 用高浓度20-羟基蜕皮甾酮溶液浸过的白桦叶片饲喂幼虫, 处理6 h, 摄入约400 μg 20-羟基蜕皮甾酮后, 幼虫停止取食; 处理12 h时表皮细胞顶膜上的微绒毛减少, 在皮细胞与旧表皮之间形成蜕皮间隙, 旧头壳从幼虫头部脱离; 处理24 h时蜕皮间隙继续增大, 旧表皮与皮细胞进一步分离, 新表皮质层开始形成; 处理36 h时皮细胞顶膜形成较短的微绒毛, 胞质区域出现数量较多的电子疏松泡, 新表皮由上表皮、 外表皮及8层左右内表皮片层组成; 处理48 h时顶膜与内表皮界限模糊, 内表皮继续合成至16层左右; 72 h时细胞内出现大面积电子疏松泡, 内表皮合成至20层左右。 处理96 h时, 与对照组相比, 皮细胞细胞器较少, 核仁周围出现小部分空白区域, 胞质区域内含物减少; 虫体发黑缩小, 即将死亡; 内表皮层数仍旧保持20层左右。对照组幼虫6-96 h虫体活跃, 正常取食, 外部观察及透射电镜结果均未显现蜕皮现象; 表皮层由上表皮、 外表皮及内表皮组成; 皮细胞顶膜微绒毛密度高; 表皮细胞分泌活动旺盛, 胞质区域细胞界限明显, 内含物丰富; 细胞器典型而且活跃; 内表皮片层随时间不断增加至50层左右。结果提示, 外源20-羟基蜕皮甾酮能够导致舞毒蛾5龄幼虫的致死性蜕皮。     

鲤鱼精子超微结构的研究

鲤鱼精子由头部,中片和尾部组成,头部的细胞核卵形,染色质致密。核中有些小空隙,空中的电子致密物质存在。中片紧连在核的后端。中片由中心粒复合体和袖套组成。中心粒复合体位于核后植入窝中,袖套一侧肥厚,一侧狭窄,袖套中有线粒体和囊泡。囊泡有二类,一类含有电子致密物质;另一类无电子致密物质。近袖套内膜处的细胞质中还存在着与内膜平行的膜,精子尾部从袖套腔中伸出。尾部的轴丝与基体相接。尾部的近核端多有许多囊泡     

Reversible plasma membrane ultrastructural changes correlated with electropermeabilization in Chinese hamster ovary cells

Chinese hamster ovary cells (CHO) grown in monolayers were permeabilized to molecules with molecular weight up to 1000 by high intensity 100 mus square wave electric field pulses. This permeability was transient and the cell viability was not affected. It was not possible for molecules with molecular weight larger than 1500 to penetrate inside the cytoplasm if lytic pulsing conditions were not used. In order to investigate the ultrastructural changes associated with this transient and limited permeabilization, cells were chemically fixed a few seconds after their pulsation and observed by electron microscopy. By scanning electron microscopy, numerous microvilli and blebs were observed almost immediately after application of the field. No other membrane changes were observed. Permeabilization of the membrane was visualized at the electron microscopic level by penetration of Ruthenium red. The appearance of osmotic pressure-dependent 'blebs' was indicative of local weakening of the plasma membrane. Most of these effects were fully reversible and disappeared within 30 min at 37 degrees C with the formation of huge polykaryons when cells were in contact before pulsing.     

Adsorption, capping, and release of a complex bacteriophage by mycoplasma cells.

By electron microscopic studies, the adsorption and release of nonlytic, cytocidal mycoplasma virus MVL3, which infects ACholeplasma laidlawii cells, have been examined. The MVL3 virion has a polyhedral head, collar, short tail, and tail fibers and contains linear double-stranded DNA. Adsorbed MVL3 virus showed a temperature-dependent clustering or capping on the mycoplasma cell membrane. During infection, a number of virus-cell membrane-related structures were observed, suggesting a general model in which MVL3-infected cells release progeny virions in membrane vesicles. These vesicles must then break down to release MVL3 particles.     

Phagocytosis of ejaculated spermatozoa.

In patients with accessory gland infections or subjects who have sperm antibodies in their semen, the presence of macrophages with phagocytic activity on ejaculated spermatozoa is significant. Light microscopy cannot certify phagocytosis because it does not give a three-dimensional view of the cells and can lead one to mistake superficial adherence of the spermatozoa to the macrophage for phagocytic activity. For that reason, scanning electron microscopy was used in this study. The samples, fixed with 2.5% glutaraldehyde in phosphate-buffered saline, were processed for observation with light microscopy (Giemsa or Papanicolaou stain) or with scanning electron microscopy (cell selection, critical point drying and paladium-platinum sputtering). With scanning electron microscopy, inactive macrophages had large membrane folds and a globular structure similar to those seen in ascites, whereas when active, they decreased in volume and developed a surface with granules or blebs. Inactive macrophages were rarely seen. A few minutes after mixing the different fractions of the ejaculate, phagocytosis reached such a level of activity that the spermatozoa partly covered the macrophages. Thus, we observed that the spermatozoa were caught by the head first in some instances but by the main-piece fragment of the tail first in other instances; very rarely were they taken by the midportion, between the head and tail. The presence in the ejaculate of macrophages with phagocytic activity on living, motile spermatozoa thus indicates that the encounter between the macrophages and spermatozoa was a result of the assemblage of components that make up the ejaculate. In this way the contributions of the prostatic gland and seminal vesicles play an important part in the spermiophagy of spermatozoa.     

瘤背石磺的精子结构

用光学显微镜和透射电子显微镜技术研究了瘤背石磺精子的结构特点,分析了其生理生态适应性以及在肺螺亚纲系统演化中的意义。瘤背石磺的精子由头部、中段和末段组成。头部由奶嘴形的顶体和长圆筒状的细胞核构成。顶体包括顶体囊和顶体构架体两部分;两者的内含物都分布均匀,电子密度稍低于细胞核;顶体基部平整,与核前端之间有一空隙,内含物电子密度极低。细胞核由电子密度高的均匀颗粒物质组成,并出现核泡;核的后端有一"杯形"的凹陷,称为核后窝。中段结构复杂,主要包括一对位于核后窝内的中心粒、轴丝、质膜、线粒体及由线粒体衍生的糖原质螺旋体、基质层和类晶体层等。末段由"9 2"结构的轴丝及外包的质膜组成,无糖原质螺旋体和其它线粒体衍生物。比较瘤背石磺精子与肺螺亚纲其它物种的精子结构,我们认为该物种的精子属于"进化型",是一类在进化地位中比基眼目高等的动物。     

Isolation and partial characterization of the plasma membrane from human spermatozoa

Ejaculated human spermatozoa were subjected to nitrogen cavitation (600 psi for ten min) to remove the plasma membrane (PM). Electron microscopic examination of the cavitated cells revealed that 33% of the PM was removed from the sperm which includes both the head and tail regions. The released membrane was separated from the cavitated cells by centrifugation followed by a discontinuous sucrose density gradient centrifugation. A single membrane population was resolved at the 1.0 M sucrose interface. Examination of the isolated membranes by electron microscopy revealed vesicles of various sizes displaying unit membrane structures. Biochemical analysis of the isolated membranes showed a threefold enrichment in the surface membrane marker 5' nucleotidase and also suggested little contamination by enzymes from the cytosol (lactate dehydrogenase) or mitochondria (cytochrome oxidase). Analytical lipid analysis of the isolated membranes revealed a 26-fold enrichment in the distribution of cholesterol, an 11-fold enrichment of phospholipids, and a cholesterol:phospholipid molar ratio of 0.83. Also found was a twofold increase in glycosphingolipids which are ubiquitous components of PM in eukaryotic cells. These data indicate that the membrane vesicles isolated after nitrogen cavitation are primarily PM.     

Histochemical and ultrastructural features of the epidermis of the land planarian Bipalium adventitium

The epidermis of the land planarian Bipalium adventitium was examined by light and electron microscopy. In all regions, the epidermis consists of a simple columnar ciliated epithelium associated with a prominent basement membrane. The epithelial cells, possessing abundant microvilli and poorly developed terminal webs, are conjoined laterally at their apical ends by septate junctions. The epidermis of the creeping sole is distinguished from that of adjoining regions by a “insunken” condition of the epithelial cells, a greater number of cilia per cell, and an absence of glandular secretions other than mucus. The insunken cells of the sole possess large glycogen disposits and attributes of metabolically active cells. Unusual intranuclear inclusions of unknown significance are also found in many of the epidermal cells in all regions. The basement membrane lacks distinct layering and consists of fine fibrils displaying a beaded appearance but no obvious cross-banding. Histochemical tests indicate that the fibrils are collagenous. In addition to mucus, secretory material found in nonsole regions includes lamellated granules and rhabdites, both stained intensely by acidic dyes. Rhabdites and the basement membrane also contain disulfide-enriched proteins. In scanning electron micrographs, the sole appears as a faint, longitudinally oriented band extending along the entire length of the animal. In all regions except the sensory border of the head, the microvilli are generally obscured by the densely arranged cilia. The sensory border consists of a row of toothlike papillae and grooves covered almost exclusively by microvilli, small club-shaped structures, and larger spherical protrusions.     

Transplasma membrane electron transport in Leishmania donovani promastigotes

Leishmania donovani promastigotes are capable of reducing certain electron acceptors with redox potential at pH 7 down to -125 mV; outside the plasma membrane promastigotes can reduce ferricyanide. Ferricyanide has been used as an artificial electron acceptor probe for studying the mechanism of transplasma membrane electron transport. Transmembrane ferricyanide reduction by L. donovani promastigotes was not inhibited by such mitochondrial inhibitors as antimycin A or cyanide, but it responded to inhibitors of glycolysis. Transmembrane ferricyanide reduction by Leishmania appears to involve a plasma membrane electron transport chain dissimilar to that of hepatocyte cells. As with other cells, transmembrane electron transport is associated with proton release, which may be involved in internal pH regulation. The Leishmania transmembrane redox system differs from that of mammalian cells in being 4-fold less sensitive to chloroquine and 12-fold more sensitive to niclosamide. Sensitivities to these drugs suggest that transplasma membrane electron transport and associated proton pumping may be targets for the drugs used against leishmaniasis.