Showing papers in "Methods in Cell Biology in 1986"

Microinjection and cytoplasmic transfer in starfish oocytes.

Abstract: Publisher Summary This chapter describes the equipment and procedures used for microinjection for which starfish oocytes are used. In this system not only the material can be injected into the cell but can also remove and transfer the constituents of the cell. This system has been applied successfully to oocytes and eggs of amphibia and mice in addition to echinoderms. Most of the system are devised, especially the elegant and simple method for controlling the injection volume by constricting the micropipet. Further modifications, including the procedure for constructing the wedge-shaped egg holder, permit to master microinjection techniques easily after a little practice. Using these procedures it is found that maturation-promoting activity occurs in a wide variety of eukaryotic cells such as mitotic cells and maturing oocytes. Maturation is induced in frog or starfish oocytes when injected with cytoplasm taken from maturing oocytes or cleaving blastomeres and with the M-phase extracts of mammalian cultured cells, including HeLa, CHO, and V-79 cells. Thus, maturation-promoting factor seems to play a general role in initiating cell division both in meiosis and in mitosis and to act non-species specifically throughout the animal kingdom.

Laboratory culture of Strongylocentrotus purpuratus adults, embryos, and larvae.

Abstract: Publisher Summary This chapter describes methods for collecting, maintaining, and culturing the purple sea urchin— Strongyfocentrotus purpuratus . The chapter presents current techniques used in raising these animals through their entire life cycle, including shedding gametes from gravid animals, culturing embryos, rearing larvae, and rearing juveniles. As the larvae must feed to complete development, the procedures for raising algae as a food source for the larvae are included. The practical procedures for induction of the fragile postmetamorphic stages and raising juveniles to adults are considered. The general protocol for rearing purple sea urchins in most cases applies to other species also. The chapter discusses various methods for management of adult sea urchins. A critical variable in maintaining adult sea urchins in the laboratory is the number of animals per unit volume of seawater and the amount of oxygen required for adequate maintenance. Care must also be exercised when packing and transporting the animals. Plastic ice chests or Styrofoam containers can be used with a layer of newspaper on the bottom to further protect tubefeet and to soak up any excess water. Shipping purple sea urchins and many other species over long distances to inland laboratories can be done successfully using the packing techniques.

Embryo dissociation, cell isolation, and cell reassociation.

Abstract: In resolving the role of cell recognition events in the process of morphogenesis it is necessary to focus on single events against a background of many complex interactions. This article presents a series of approaches that are designed to do just that. It should be noted that with simplification there is a danger of oversight. Nevertheless, in learning about mechanisms of cellular movement at gastrulation we have found that cell separation techniques, simplified adhesion assays, and predictable antibody activities are helpful for approaching the complex mechanisms of morphogenesis.

Gonad-stimulating and maturation-inducing substance

Abstract: Publisher Summary This chapter describes methods for preparing and assaying gonad-stimulating substance (GSS) and maturation-inducing substance (MIS). These methods are derived from experience with selected starfish. In principle, if not in specifics, they may be useful for additional species of starfish and for studying GSS and MIS in other classes of echinoderms. Although all nervous tissues in starfish probably contain GSS, only the radial nerves are easy to dissect and handle for extracting GSS. Radial nerves are located along the ambulacral groove on the oral surface of each arm. To separate radial nerves from a soft-bodied starfish such as Asrerias arnurensis, the oral surface of the arms is dissected by cutting along the rows of tube feet with scissor. To obtain large amounts of MIS, fresh ovaries (200 mg/ml) are incubated in seawater containing GSS (500 μg dry nerve Eq/ml) for 6 hours (20°C). The medium is filtered through a mesh to remove ovarian walls and then centrifuged to remove eggs (3000 rpm for 10 minutes). Identification and use of GSS and MIS in many other animals will enable more suitable materials to be used leading to a deeper understanding of spawning and oocyte maturation.

Handling, labeling, and fractionating sea urchin spermatozoa.

Abstract: Publisher Summary This chapter describes methods employ in the research on the biochemistry of sperm–egg interaction. These methods devised for this are handling, labeling, and fractionating sea urchin spermatozoa. A pure sperm suspension can be prepared comprising composition of semen and removal of pigmented phagocytes and with method for washing sperm. Heavy metal chelators, amino acids, and peptides prolong life of sperm. A simple method for determining sperm concentration is to measure the turbidity of a sperm suspension with a spectrophotometer and relating the value to a standard curve constructed by hemocytometer counts. The sperm components are labeled by radioiodination of membrane glycoproteins, labeling with [ 125 I]diiodofluorescein isothiocyanate (IFC), labeling the surface with lectins and antibodies, and labeling sperm with radiophosphate. The isolation of sperm components comprises separation of head from flagellum, isolation of components of the sperm head, and isolation of the plasma membrane.

Techniques for observing living gametes and embryos.

Abstract: Publisher Summary This chapter describes various ways of handling relatively small numbers of echinoid gametes and embryos and of microscopically observing their physiology and development. The chapter focuses on the ways to keep the embryos healthy for extended periods of observations. For microscopic observations, the optically clear cells are preferred. Thus, the use of Lytechinus variegatus and Echinarachnius parma is emphasized, whose eggs are the clearest. To obtain healthy embryos, it is important to have healthy adults. Therefore, the discussion on the adult animals is focused. The chapter outlines the procedure that improves the success of fertilization and the viability of the cultures. Video imaging has many advantages for the researcher studying cell structure and physiology. Using a SIT video camera, it follows individual injected embryos from fourth division until midpluteus stages and observes the fate of the injected cell and its progeny at various stages in between. Even at the later stages, clearly identifies the descendants of a single, microinjected cell.

Cytology and immunocytochemistry.

Abstract: Publisher Summary This chapter describes techniques that worked out primarily for use with Strongylocentrotus purpuratus and Lytechinus pictus . The chapter discusses whole mounts of fixed and embedded material, sectioned and stained material for bright-field light microscopy, and indirect immunofluorescence microscopy and other fluorochrome probes. Whole mounts are useful for establishing a developmental timetable, where paired samples are taken for other studies. They do not provide good resolution, however are adequate to establish the timing of such developmental markers as pronuclear fusion, streak stage, nuclear membrane breakdown, furrowing, and in later stages, cell number, cilia formation, and gastrulation. A more time-consuming method for monitoring stages of development, but one which provides considerably more accuracy, utilizes stained sections of epoxy-embedded material. The success of immunofluorescence microscopy will depend largely on the quality of the antibodies that are used—that is, the specificity and affinity of the antibody for the antigen, the titer of the elicited antibody, and nonspecific staining. The fluorochrome probes including bisbenzimide (hoechst 33258) stain for DNA, NBD-phallacidin and rhodamine-phalloidin stains for actin are discussed.

Nuclei and chromosomal proteins.

Abstract: Publisher Summary This chapter explains some of the procedures that employed in the isolation of nuclei and nuclear proteins from the sea urchin embryo. Most of our work has focused on pronuclear proteins, especially those of the male pronuclei. The chapter focuses on techniques that developed especially for their study. Most of these techniques can be adapted for, or applied directly to, nuclei from embryos and other sources. In studying male pronuclei, two problems common to all nuclear isolation procedures occur in exaggerated form: those of purity and yield. Purity and yield tend to be low because of the low nuclear/cytoplasmic protein mass ratio characteristic of fertilized eggs. These problems in the sea urchin are solved by using moderately polyspermic eggs, thereby increasing the male pronucleus/cytoplasm ratio. Polyspermy also renders the contribution of the female pronucleus to the isolate quantitatively insignificant. Following nuclear isolation further purification is often necessary to separate individual molecular species and to characterize the small quantities of protein obtainable from embryonic sources. For these purposes a two-dimensional gel electrophoretic system is developed that optimally separates histones from contaminants and from each other and have designed an apparatus that allows quantitative recovery of proteins from fixed and stained gel spots. The proteins may then be subjected to various forms of microanalysis.

Isolation of embryonic cilia and sperm flagella.

Abstract: Publisher Summary Isolated cilia are useful for developmental studies concerning gene expression, organelle morphogenesis, and kinetic/length determination Studies with protein synthesis inhibitors indicate that the cilia are assembled mainly from preexisting protein pools Even the regeneration of cilia can take place quite normally in the presence of inhibitors of protein synthesis With an adequate supply of healthy sea urchin embryos and the most basic technical skills, the isolation of embryonic cilia, particularly using successive regenerations, is relatively easy For biochemical comparison of microtubule-based motile systems, sperm flagella offer an abundant source of material and involve equally simple isolation techniques This chapter discusses isolation method and fractionation method The chapter outlines presupposes that the worker is capable of fertilizing eggs without producing polyspermy and is able to grow the embryos through hatching without significant cell death or loss of synchrony and obtain sperm by potassium chloride (KCl) injection, electrical stimulation, or excision of gonads

Purification and properties of the egg plasma membrane.

Abstract: Publisher Summary The chapter focuses on the preparation of plasma membranes from unfertilized and fertilized eggs that are low in contamination by other organelles and that, in vesicle form, are amenable to biochemical analysis. The chapter describes the details of these methods and presents some of the ways that these membrane preparations can be used. The preparation of plasma membranes from unfertilized eggs is greatly facilitated by the fact that large sheets of plasma membranes with associated cortical vesicles (cell surface complex) can be isolated quickly and in good yield. The cortical vesicles can then be released from the plasma membrane sheets, which are then purified by differential and density gradient centrifugation. This method yields sheets of plasma membrane that retain many vitelline layer components including the sperm receptors. The egg plasma membranes including morphology, enzymatic activity of the egg plasma membrane, and protein composition are characterized. The methods presented in the chapter allow the preparation of useful quantities of plasma membranes from the sea urchin egg. These membranes exhibit enzyme activities and at least some of the transport properties that are characteristic of other eukaryotic plasma membranes. These preparations are routinely used to study fertilization-dependent changes in tyrosine protein kinase activity and they should be useful for other enzymes as well.

Egg and embryonic extracellular coats: isolation and purification.

Abstract: Publisher Summary This chapter focuses primarily on the methods available for analysis of fertilization membrane assembly—namely, the isolation of precursors, individual components, and intermediates of the assembled fertilization membrane. Isolation of hyalin, the major protein component of the hyaline layer, readily follows as a useful by-product of these methods. All of the methods described pertain (primarily) to Strongylocentrotus purpuratus eggs and embryos, the species for which fertilization membrane assembly has been most studied. The chapter reviews the methods that are valuable for examining modifications in egg surface coats after fertilization. The chapter discusses a schematic diagram of the several fertilization membrane components and the relationships between them. The applicability, advantages, and disadvantages of the method are also discussed. The chapter demonstrates the purification of proteins from fertilization product, and how the protocols for isolating individual proteins might be integrated to purify several components from the same starting material.

Manipulative methods for analyzing embryogenesis.

Abstract: Publisher Summary This chapter describes a number of manipulative methods adopted to analyze the egg cells and embryos of the sea urchin Hemicentrotus pulcherrimus and the starfish Asterina pectinifera . With minor modifications, they will be applicable to other species. By using egg fragments, the possible roles for the nucleus in cytoplasmic activities or, in turn, possible roles for cytoplasmic materials in embryogenesis are examined. There are two ways to obtain egg fragments: manual bisection and centrifugal separation. Manual bisection enables to divide eggs in any desired direction so that single egg fragment can be compared with its complementary partner. On the other hand, centrifugal separation makes large enough numbers of nonnucleate and nucleate egg fragments for biochemical analyses. The chapter describes how horseradish peroxidase (HRP) is injected into blastomeres of starfish embryos through the intact fertilization membrane. Chromosome preparations of starfish embryos are explained.

Scanning electron microscopy of embryos.

Abstract: Publisher Summary This chapter provides descriptions of wet chemical, alcohol dehydration and critical point-drying methods that developed for routine preparation of sea urchin embryos for scanning electron microscopy (SEM) analysis in the secondary electron mode. Therefore, artifacts of specimen preparation and operational parameters of the SEM scope are reviewed. After the basic procedures of preparing specimens and the morphological information derived from SEM secondary electron images are obtained, then is the time to use SEM instrumentation and techniques for further bridging the gap between light microscopy (LM) and transmission electron microscopy (TEM) analyses. In particular, secondary electron images of the labeling of cell surfaces through markers such as latex spheres and colloidal gold coupled with improved techniques for preserving the integrity of hydrated molecules can vastly improve the detection and localization of a number of developmentally interesting macromolecules. Finally, backscatter electron imaging of structures differentially “stained” with various heavy metals in addition to colloidal gold markers offer unexplored opportunities. The main limits of these opportunities are inertia, time, and imagination.

Rapid repetitive microinjection

Abstract: This article describes methods to inject large numbers of echinoid eggs and zygotes with globin mRNA and label them with tritiated amino acids. The products can be quantitatively analyzed to determine the amounts of peptide synthesis. The injections can be performed quickly and require relatively simple and inexpensive equipment. Although I designed my methods to inject large numbers of echinoid eggs and zygotes, these methods should also be useful for injecting many different molecules and ions into smaller numbers of cells as well as into a variety of cell types.

Tubulin-containing structures.

Abstract: Publisher Summary The chapter describes methods for the purification of echinoderm egg and spindle tubulins, microtubule-associated proteins, mitotic organizing centers, and, briefly, cilia and flagella. The chapter discusses some of the relevant aspects of the biochemistry of echinoderm tubulin and microtubule reassembly in vitro . Many of the components presumed necessary for the assembly and organization of mitotic spindle microtubules are present in the unfertilized sea urchin egg, including assembly competent tubulin, microtubule-associated proteins, and latent mitotic organizing centers. Despite the storage of microtubule components in the unfertilized egg, there are few, if any, assembled microtubules. Fertilization triggers the assembly of microtubules, and many other events such as the alkalinization of the Cytoplasm from pH 6.80 to 7.30. The increase in intracellular pH could release an inhibitor molecule or protein from egg tubulin, allowing assembly to occur in the presence of microtubule-associated proteins and mitotic organizing centers. The understanding of mitosis and microtubule function will benefit from promising new research and an ability to isolate and characterize these tubulin-containing structures in vitro .

Chapter 3 Sperm Motility

Abstract: Publisher Summary This chapter comprises of experimental preparations, and observation and measurement of sperm motility. The Experimental preparations include sperm collection, live spermatozoa and demembranated spermatozoa. Most of the available data on sperm motility have been obtained from spermatozoa swimming near surfaces. Microscopic observation of spermatozoa swimming in the interior of a microscope slide chamber, away from surfaces, is difficult. Although photographic methods can provide very detailed information about flagellar bending, they sample relatively small numbers of spermatozoa in a population. It is desirable to have that method that measured average motility parameters of a larger sperm population and of a population of spermatozoa that is not confined to the vicinity of a surface. Such a method would be particularly useful for comparing motility with parameters such as metabolic rate. As 90% or more of sperm energy metabolism is normally coupled to motility, measurements of the oxidative metabolism of live spermatozoa or the rate of ATP dephosphorylation by demembranated spermatozoa are important adjuncts of sperm motility studies. In both cases, measurements should be made on suspensions with relatively low sperm density if it is also desired to measure motility parameters in samples taken from the suspensions during the measurements, without modifying the sperm environment by additional dilution.

The compression method for determining the surface force.

Abstract: Publisher Summary This chapter discusses a compression method for determining the surface force. When a spherical egg with the initial diameter Z 0 is compressed between parallel plates with a constant load F, the egg will be gradually flattened over several minutes until it attains an equilibrium thickness Z. The ratio Z/Z 0 serves as an estimate of the stiffness of the egg. The flattening of course causes a stretching of the egg surface and global deformation of the bulk endoplasm. This compression method does not by itself distinguish between the stiffness of the cortical layer and possible contributions from structures in the inner cytoplasm. The measurements of the viscoelasticity of the cytoplasm of sea urchin eggs indicate that the inner cytoplasm may temporarily resist deformation when subjected to external forces. The value of Z/Z 0 represents a measure of the mechanical property, or the “stiffness,” of the egg cortex. Changes in this value of Z/Z 0 are taken as indicators of certain activities of egg cytoplasm in various conditions. The measured stiffness affords clues to the nature of possible structures that support the integrity of the egg cortex. The chapter also discusses calculation of the surface force and the weight loading method, and concludes with some critical comments on the methods.

Cytological techniques for the study of larval echinoids with notes on methods for inducing metamorphosis.

Abstract: Publisher Summary This chapter presents two techniques that are useful in identifying structure and organization—monoclonal antibodies against tissue-specific antigens in sea urchin embryos and visualization of larval nerves with glyoxilic acid-induced fluorescence. These techniques have provided reliable cytological markers that have assisted in determining aspects of the origin and fate of certain tissues of larval echinoids. It is found that a whole-mount immunofluorescent technique valuable for identifying clones secreting tissue-specific antibodies. It is simple to prepare and allows positive, three-dimensional localization of the cells without serial sectioning. Modifications of the basic procedures are primarily in the preparation of the immunogen and in screening techniques for immunofluorescent localization of cell surface, tissue-specific antigens on whole-mount larvae. As studies of the fate of larval tissues often require a detailed analysis of the morphogenetic movements that the larva undergoes during metamorphosis, therefore several methods for the induction of metamorphosis in echinoids are included—namely, the extraction and electrical methods.

Simple methods and devices for handling echinoderm eggs.

Abstract: Publisher Summary This chapter explores that all experience with echinoderm eggs is related to investigations on cleavage mechanisms. Techniques used in handling eggs and in micromanipulation acquired by personal trial and error. All investigations have involved the use of inverted microscopes that provide easy access to the operation chamber and permit simple solutions to the problems of adequate working distance and evaporation of the medium. The chapter describes several simple methods and easily made devices that have proved useful in experimentation on echinoderm eggs. Equipment for making instruments includes alcohol burner and microforge. The chapter discusses fabrication and care of instruments, which includes cutting glass, cements, and ultrasonic cleaning. The devices discussed include braking pipet, operating chamber, silicone rubber capillary and micromanipulator. Beyond providing detailed instructions for a few methods, it is just as important to stress that simple, direct methods can still yield valuable data in important areas of experimentation.

Induced fusion of echinoderm oocytes and eggs.

Abstract: Publisher Summary The chapter reviews the methods of induced fusion of echinoderm oocytes and eggs. It has shown that the procedures leading to the fusion of oocytes or eggs do not affect the normal condition of the cells: they retained the capacity for maturation, fertilization, and subsequent development. The use of the induced fusion method in studies on the echinoderm oocytes and eggs makes it possible to obtain mass quantities of oocytes and eggs combined at different developmental stages or to alter (qualitatively or quantitatively) the nucleocytoplasmic ratio. The technique of induced cell fusion calls for the solution of three problems: (1) the removal of all envelopes peripheral to the plasma membrane—that is, the removal of the follicular envelope and vitelline membrane; (2) the agglutination of the oocytes; and (3) the fusion of the apposed membranes. Therefore, they are conducted at temperatures of 13-2O˚C, in an air-conditioned room when necessary, with the use of artificial seawater. The possibility of egg fusion in echinoderms is corroborated by the spontaneous and rare appearance of fused sea urchin eggs after treatments designed to remove the fertilization membrane.

Determination of mechanical properties of the egg surface by elastimetry

Abstract: Publisher Summary Elastimetry is a method for measuring mechanical properties of the cell surface based on the relation between its deformation and a negative pressure applied to a part of the cell surface through a micropipet Elastimetry is used for measuring mechanical properties of the cell surface, especially in echinoderm and amphibian eggs and in blood cells In elastimetry, a micropipet whose tip is cut at right angles to the micropipet axis is brought into contact with the cell surface, and the deformation of the cell surface and the negative pressure applied through the micropipet are measured Because the deformation is restricted to a part of the cell surface, this method is suitable for measurement of cells with mechanical properties that differ at different regions of the cell surface A typical experimental set-up for elastometry is discussed To compare mechanical properties of the cell surface at two regions of the cell, it is desirable to use a pair of micropipets with identical sizes, as the stiffness depends on the size of the micropipe Mechanical properties—namely, stiffness and surface force of the cell surface are discussed

The protein synthetic machinery: ribosomes and cell-free systems.

Abstract: Publisher Summary This chapter discusses several approaches that can be used in analyzing protein synthesis machinery. In vivo, the kinetics of reactions can be monitored; the polyribosome sizes and distribution can be measured; and the cells can be perturbed by changing internal ion concentration with external treatments such as ammonia or acetate that change internal pH, or calcium ionophores that change internal calcium concentrations. This method gives insight into the ionic events responsible for the activation of protein synthesis in fertilized eggs. Advantages of using in vitro systems are that individual components can be tested, and specific responses to a large variety of inhibitors of protein synthesis that are not membrane soluble can be measured. A goal of in vitro studies is to mimic both the repression of protein synthesis in eggs, and the derepression of the inhibited components by using only the discrete molecules and ions involved in the repression and activation processes.

Determination of mechanical properties of the egg by the sessile drop method.

Abstract: Publisher Summary This chapter discusses the mechanical properties of the egg by the sessile drop method. The stiffness expressed by the different parameters depends on various mechanical properties of the cell components, for example, the surface force, rigidity of the cortex, intracellular pressure, or rigidity of endoplasmic structures. The surface force is determined from the degree of deformation of the egg under a gravitational or centrifugal force, the density difference between the egg protoplasm and the surrounding medium, the size of the egg, and the magnitude of the gravitational or centrifugal acceleration, using relationships obtained from the form of a theoretical sessile drop. A simple and accurate method to determine the density of the egg protoplasm is by the “isopycnotic method”. The chapter discusses possible errors and limitations of the sessile drop method in determining the surface force of the sea urchin eggs. It stated that neglecting the rigidity of the endoplasm may not introduce serious errors in surface force determination in unfertilized sea urchin eggs. In fertilized eggs, however, the errors may be larger because structures having considerable rigidity such as the sperm aster or mitotic apparatus are formed in the endoplasm. It is supposed that the density of the protoplasm is practically uniform in echinoderm eggs because eggs do not display any orientations in gravitational fields.

Ion measurements in sea urchin sperm.

Abstract: Publisher Summary This chapter discusses methods used to measure ion changes important to activation of sea urchin sperm motility and the acrosome reaction. The methods used to measure pH i , ion fluxes, and membrane potentials in sea urchin sperm are described. Methods available for measurement of pH i include uptake of radiolabeled weak acids and bases, 31 P-nuclear magnetic resonance, and incorporation of molecular probes such as carboxyfluorescein that undergo spectral or fluorescent shifts with changes in pH i . Simple ionic manipulation, regulation of pH i , and [Ca 2+ ] are of fundamental importance to sperm physiology. pH i is discussed as it is an important regulatory component of sperm motility and the acrosome reaction. pH i changes in sea urchin sperm correlate with or are linked with other cation movements also. In particular, a Na + /H + exchange is a component of the sperm plasma membrane that can either raise or lower the pH i . Induction of the acrosome reaction results in multiple changes in the sperm cation composition. Na + and Ca 2+ enter the sperm and K + (and H + ) exit. Of these Ca 2+ entry is a crucial requirement for the acrosome reaction regardless of the method used to initiate it.

Components of the actin-based cytoskeleton

Abstract: Publisher Summary This chapter focuses on the methods that developed for the extraction and study of proteins of the actin-based cytoskeleton, using as material primarily the gametes of the Hawaiian sea urchin, Tripneustes gratilla . It is one of the more common local species and also offers the advantage of large size, so that 10-20 ml of gametes can be obtained from a ripe animal. The behavior of extracts is undoubtedly temperature dependent, so appropriate adjustments of temperature when working with other species will have to be made. The chapter focuses on the cytoplasmic extract as a model system for studying cytoplasmic behavior. The detailed procedures described in the chapter include the preparation of extracts, the recovery of actin, myosin, and several other actin-associated proteins using procedures based on the specific properties of these extracts, methods for the study of the interaction of these components in vitro , and the relation of these reactions to cellular events.

Electrophoresis of eggs and blastomeres.

Abstract: Publisher Summary This chapter describes the methods to make a rectangular, horizontal type of cell electrophoresis apparatus and to measure the electrophoretic mobility of the eggs and blastomeres of sea urchins. The apparatus discussed are electrophoresis chamber, circuit and electrode, and circulation system for constant temperature. Using the electrophoresis chamber, the electrophoretic mobilities of the micromere-and mesomere-derived cells of sea urchin embryos are examined during their development, showing that the negative charges of the cell surface increase during the migration as primary mesenchyme cells of the micromere-derived cells in vitro . Measurements of the surface charges of cell organelles, such as nuclei, are also possible using an electrophoretic method. As reviewed briefly, cell electrophoresis is a unique method for detecting changes that occur at the outer surface of living cells and organelles. Although this method is limited by the fact that the only property it detects is electric charge or potential, it seems to provide a useful tool for analyzing cell surface architecture when combined with other methods that modify or dissect the molecules on the cell surfaces.