Synthesis and accumulation of ribosomes in individual cells of the female gametophyte of maize during its development

Summary The pattern of synthesis of ribosomes during three stages of development of the female gametophyte of maize has been studied by in situ hybridization using a ribosomal RNA probe. Changes in volume of individual cells of the embryo sac during its maturation have been determined by confocal microscopy. These data have permitted us to calculate the relative numbers of ribosomes in the cells of the embryo sac at different stages of their maturation. The egg apparatus and the central cell at all stages of development contain several fold greater numbers of ribosomes than are present in the antipodal cells or cells of the surrounding nucellus. The accumulation of ribosomes during embryo sac maturation appears to proceed at a constant and high rate, with the rate being highest in the developing central cell.     

Cellular localization of a pollen-specific mRNA by in situ hybridization and confocal laser scanning microscopy

Summary The application of confocal laser scanning microscopy together with in situ hybridization experiments in tobacco pollen enabled a detailed localization of a pollen-specific mRNA. The three-dimensional distribution of this specific mRNA over the whole pollen grain was reconstructed by means of optical sections of one specimen.     

Structural characteristics of the mature embryo sac of Camellia oleifera

The ovule is the most important reproductive organ in the pistil of phanerogamae. Camellia oleifera (Theaceae) is an important woody plant producing edible oil in southern China, and its embryo sac structure has a positive effect on seed breeding. In this study, the microstructure, ultrastructure and three‐dimensional structure of the ovule and embryo sac of C. oleifera were observed and described based on a combination of advanced microscopy techniques (SEM, TEM, CLSM). The ovule comprises the inner and outer integument. Large quantities of secretions in the micropylar canal exit and may participate in the guidance of the entry of the pollen tube into the embryo sac. The synergids have a dense cytoplasm, abundant organelles, and strong polarity. Little cytoplasm is present in the egg cell, yet there are many vacuoles. The center of the cell is taken up by a large vacuole, and the cytoplasm is pushed towards the edges to form obvious cytoplasmic cords. The two polar nuclei are large and conspicuous. The antipodal cells degenerate to fulfill a nutritional function.     

Ultrastructural in situ hybridization: A review of technical aspects

Detection of nucleic acid sequence at the ultrastructural level has allowed us to better understand the expression of genes in some fields of application in cell biology. In situ hybridization at the ultrastructural level can be carried out using three different methods: on vibratome sections before embedding in epoxy resin, on ultrathin frozen section, or on ultrathin section of tissue embedded in hydrophilic resin such as Lowicryl. Before starting the detection of nucleic acid sequences at the electron microscope level, the experimenter has to choose various parameters: the type of tissue fixation, the probe and its label, and the in situ hybridization method, depending on the sensitivity, the resolution and the ultrastructural preservation required. This review of technical aspects, by describing the different methods of ultrastructural in situ hybridization, will help the experimenter to optimize each step of the hybridization procedure.     

Localization of the T-DNA on marker chromosomes in transformed tobacco cells by in situ hybridization

Summary Chromosome and molecular analyses were conducted on tobacco cells which had been transformed by the T-DNA of the Ti-plasmid. These analyses showed that there were specific chromosome rearrangements in the transformed cells (marker chromosomes). There was a positive correlation between the number of marker chromosomes per cell and the oncogenic potential of the transformed cells. However, we show, using the Southern hybridization method, that the T_L fragment of T-DNA, but not the T_R, clearly hybridizes with nuclear DNA. In situ hybridization was used to locate the insertion site of T-DNA: the hybridization signal was found on a small metacentric chromosome. This chromosome may occur single or translocated onto other chromosomes, to make marker chromosomes. Thus, by locating the T-DNA, we have confirmed the correlation between the marker chromosomes and the oncogenic potential.     

原位杂交检测microRNA-205在乳腺癌中的表达

目的探讨microRNA-205表达与乳腺恶性病变的关系。方法乳腺疾病及癌组织芯片原位杂交分析microRNA-205的表达;实时定量RT-PCR方法检测正常乳腺细胞株、恶性程度不同的乳腺癌细胞株中microRNA-205的表达。结果原位杂交分析显示,36例正常与良性乳腺病变中,33例(91.67%)表达阳性;36例乳腺癌中,23例(63.89%)表达阳性。microRNA-205的表达在乳腺正常与良性病变中的表达较恶性病变中高且有统计学差异(P=0.011),但与乳腺癌TNM分期、临床分期无关(P0.05)。实时定量RT-PCR结果显示,四个高度恶性乳腺癌细胞株(MDA-MB-231、HS578T、BT549和SUM159PT)中microRNA-205的表达较永生化正常乳腺上皮细胞株MCF10A和四个低度恶性细胞株(MDA-MB-468、T-47D、ZR-75-1和SKBR3)中为低(P0.05)。结论原位杂交适用于microRNA-205的表达分析;组织芯片标本原位杂交与乳腺细胞株实时定量RT-PCR分析结果提示,microRNA-205可能参与了乳腺癌的发生、发展,并随着乳腺癌的演进呈下调趋势。     

Changes in the morphology of ribosomes in uterine epithelial cells under various hormonal conditions

Summary The ultrastructural morphology of free cytoplasmic ribosomes of rat uterine epithelial cells was studied during diestrus, estrus, after ovariectomy, and after estradiol-17 administration to rats that were ovariectomized 1 to 25 weeks before hormone treatment. A change in size and contrast of ribosomes was observed concomitant with a transition from pre-existing monosomes to polysomes depending on the dose of estradiol and its route of application. In 3 weeks ovariectomized rats these changes in ribosomal granules take place at approximately the same time (30–45 min) when synthesis of induced protein was described biochemically. The morphological events after estradiol administration are discussed with respect to a primary site of estrogen action.The author wishes to thank Professor E.J. Plotz for continued support, Professors P. Gedigk and V. Totovic for electron microscope facilities in the Dept. of Pathology, Professor E.C. Roosen-Runge for valuable suggestions, and Miss S. Vosberg and Mrs. M. Przybilka for expert technical assistence.This study was supported by the Deutsche Forschungsgemeinschaft     

Lipid droplets in the visceral yolk sac endodermal cells of the postimplantation rat embryo: Application of malachite green-glutaraldehyde fixative

Summary The ultrastructure of the visceral yolk sac (VYS) of the rat embryo at day 9.5 of gestation was examined after fixation with either Karnovsky's glutaraldehyde-paraformaldehyde solution or malachite green-containing glutaraldehyde (MGA) solution. Fixation with MGA retained homogeneously electron-dense droplets in the cytoplasm and the nucleus of endodermal cells, both of which were lost in the specimens prepared by Karnovsky's fixation method. The cytoplasmic MGA-positive droplets were frequently associated with other cytoplasmic organelles such as rough endoplasmic reticulum, mitochondria and membrane-delineated inclusion bodies, but these cytoplasmic organelles never incorporated MGA-positive materials, whereas Golgi apparatus contained intracisternal MGA-positive droplets. Extracellular MGA-positive droplets were also encountered at the apical surface of endodermal cells and in the intercellular space between endodermal cells and the underlying mesodermal cells. These MGA-positive droplets were considered to be lipid in nature, and their origin in the endodermal cells of VYS is discussed.     

玉米近缘种中玉米着丝粒序列的FISH检测

为分析玉米着丝粒卫星DNA(CentC)和着丝粒反转录转座子(CRM)在玉米种的亚种及近缘种中的保守性,采用双色荧光原位杂交检测了这两种重复序列在墨西哥玉米、二倍体多年生类玉米、多年生类玉米、摩擦禾、薏苡、高粱中的存在和分布。CentC、CRM探针在墨西哥玉米、二倍体多年生类玉米和多年生类玉米的所有染色体的着丝粒区产生了强或较强的杂交信号, 而且不同染色体的杂交信号的强度存在差异, 表明两种玉米着丝粒重复序列在不同着丝粒中的数量不同; 此外, 部分着丝粒中的CentC和CRM信号的强度存在差异, 不完全重叠。CentC、CRM探针仅在摩擦禾的多数染色体的着丝粒区产生了弱的杂交信号。在薏苡和高粱中仅测检到主要分布在着丝粒区的较强或强的CRM信号。这些结果表明, CentC在玉米种的亚种间及玉蜀黍属的物种间高度保守, 在与玉蜀黍属亲缘关系最近的摩擦禾属物种中也具有较高的保守性; CRM在与玉蜀黍属亲缘关系较近和较远的禾本科种属中都具有保守性。     

Callose in the walls of mature embryo sac ofTorenia fournieri

Summary During the course of a fluorescence microscopic investigation on the extra-ovular micropylar portion of the embryo sacs ofTorenia fournieri Lind. (Scrophulariaceae) a callosic wall was found which surrounded it almost completely until the time of anthesis. In addition, the walls of young synergids and the filiform apparatus also showed callosic fluorescence. Treatments with PAS reaction revealed a PAS-positive substance filling up the locular cavity. Our attempts to induce fluorochromasia by employing fluorescein diacetate failed, indicating the low permeability of the callosic wall around the embryo sac. It is assumed that the callose wall around the embryo sac isolates the latter from the contents of the locular cavity whereas the callose in the synergid walls may represent an intermediate stage in the maturation of these walls; the filiform apparatus is mainly composed of callose.     

Development of ovule,embryo sac,and endosperm in Dipterostemon and Dichelostemma (Alliaceae) relative to taxonomy

Dipterostemon capitatus and all species of Dichelostemma have the following characters in common: the ovule is anatropous and bitegmic; the nucellar epidermis is penetrated by the embryo sac; the remaining chalazal part of the nucellus expands, partly due to periclinal divisions in its epidermis; the micropyle is formed by the inner integument only; parietal cells are present; the embryo sac develops according to the Polygonum type; the endosperm is nuclear; after fertilization, the cells of the inner integument enlarge greatly. One difference between the genera is that the inner integument is two cells thick in Dipterostemon, but five to seven cells thick in all Dichelostemma species. In Dipterostemon, embryogenesis is of the Asterad type, and the formation of walls in the endosperm occurs much as in wheat. The absence of variation in embryological characters among the species of Dichelostemma strongly supports the view that these species all belong in one genus, despite differences in gross morphology. Dichelostemma is very closely related to Brodiaea, since species of these two genera show identical embryology. Dipterostemon must be retained as a genus because a deviating inner integument adds to uniqueness in gross morphology. Dipterostemon, Dichelostemma, and Brodiaea, are embryologically quite different from Triteleia. Neither Dipterostemon nor Dichelostemma show any close affinity with Allium.     

A methacrylate embedding procedure developed for immunolocalization on plant tissues is also compatible with in situ hybridization.

A recently described method that uses methacrylate embedding of aldehyde fixed plant tissues allows the immunolabelling of a range of antigens (Baskin et al. 1992). We have tested whether the same embedding procedure is also compatible with in situ hybridization. For this purpose we have used 2- 5 μm sections of methacrylate embedded plantlets of Arabidopsis thaliana. After removal of the resin the sections were prepared for in situ hybridization following standard procedures. Three different digoxygenin (dig)-labelled probes were used, recognizing RNAs coding for the chlorophyll a/b binding protein cab-140, the β-tubulin tub5 and meri a member of the meri-5 family. Each of the probes shows the labelling pattern expected from the literature. Moreover, the method allows a good structural preservation of very fragile tissues, in contrast to paraffin embedding. We conclude that methacrylate embedding, allowing both immunolabelling and in situ hybridization with high resolution and structural preservation, offers a high potential for the functional analysis of genes and proteins in plant development. This is especially true for Arabidopsis thaliana, a widely used model species where it seems to be the method of choice.     

Expression of the gene encoding the PR-like protein PRms in germinating maize embryos

Summary The PRms protein is a pathogenesis-related (PR)-like protein whose mRNA accumulates during germination of maize seeds. Expression of the PRms gene is induced after infection of maize seeds with the fungus Fusarium moniliforme. To further our investigations on the expression of the PRms gene we examined the accumulation of PRms mRNA in different tissues of maize seedlings infected with E. moniliforme and studied the effect of fungal elicitors, the mycotoxin moniliformin, the hormone gibberellic acid, and specific chemical agents. Our results indicate that fungal infection, and treatment either with fungal elicitors or with moniliformin, a mycotoxin produced by F. monilforme, increase the steady-state level of PRms mRNA. PRms mRNA accumulation is also stimulated by the application of the hormone gibberellic acid or by treatment with silver nitrate, whereas acetylsalicylic acid has no effect. In situ RNA hybridization in isolated germinating embryo sections demonstrates that the PRms gene is expressed in the scutellum, particularly in a group of inner cells, and in the epithelium lying at the interface of the scutellum and the endosperm. The pattern of expression of the PRms gene closely resembles that found for hydrolytic enzymes, being confined to the scutellum and the aleurone layer of the germinating maize seed. Our results suggest that the PRms protein has a function during the normal process of seed germination that has become adapted to serve among the defence mechanisms induced in response to pathogens during maize seed germination.     

Fatty acid esterification in the yolk sac membrane of the avian embryo

The transfer of lipid from the yolk to the avian embryo is mediated by the yolk sac membrane (YSM). Some, but not all, of the published morphological evidence supports the view that the lipid undergoes a cycle of hydrolysis and re-esterification during translocation across the YSM. The present study aims to test this view by investigating the capacity of the YSM to perform esterification of free fatty acids to form acyl-lipids. YSM pieces (area vasculosa), obtained from the chicken embryo at day 10 of development, were incubated in vitro in medium containing [¹⁴C]-palmitic acid. Radioactivity was rapidly incorporated into the tissue lipid indicating a high capacity for esterification. The incorporation was linear with time during the 1-h incubation. Approximately 84% of the incorporated label was recovered in triacylglycerol, 12% was incorporated into phospholipid and less than 1% was detected in cholesteryl ester. [¹⁴C]-palmitic acid was incorporated primarily at the sn-1/3 positions in the triacylglycerol molecule and at the sn-1 position of phospholipid. The incorporation of label into tissue pieces obtained from the non-vascularized peripheral region of the YSM (area vitellina) was much more limited than that observed for the area vasculosa. The results support the hypothesis that yolk lipid is hydrolyzed and re-esterified during transfer across the YSM.Abbreviations YSM yolk sac membrane - VLDL very-low density lipoprotein Communicated by G. Heldmaier     

An in situ hybridization protocol for planarian embryos: monitoring myosin heavy chain gene expression

The monitoring of gene expression is fundamental for understanding developmental biology. Here we report a successful experimental protocol for in situ hybridization in both whole-mount and sectioned planarian embryos. Conventional in situ hybridization techniques in developmental biology are used on whole-mount preparations. However, given that the inherent lack of external morphological markers in planarian embryos hinders the proper interpretation of gene expression data in whole-mount preparations, here we used sectioned material. We discuss the advantages of sectioned versus whole-mount preparations, namely, better probe penetration, improved tissue preservation, and the possibility to interpret gene expression in relation to internal morphological markers such as the epidermis, the embryonic and definitive pharynges, and the gastrodermis. Optimal fixatives and embedding methods for sectioning are also discussed. A. Cardona and J. Fernández have contributed equally to this work.