Robert H. Spitzer

Multiple effects of colchicine on oogenesis in Drosophila: Induced sterility and switch of potential oocyte to nurse-cell developmental pathway

SummaryAdult female fruitflies exposed to colchicine admixed to the culture medium show a series of dosage-related abnormalities that affect oogenesis and may induce sterility. Among the effects observed were decreased fecundity and hatchability of laid eggs, formation of oocytes lacking chorionic appendages, abnormal distribution and diminution in number of yolk spheres, inhibition of oocyte growth and abnormally located oocyte nuclei. Potentially the most significant effect was the development of egg chambers which contained the normal complement of 16 cells but in which all the cells had the nuclear morphology of nurse cells. The approach provides for the first time an experimental means to divert a potential oocyte into the developmental pathway of the nurse cell in a wild-type fly, and hence should be helpful in the elucidation of factors which control oocyte and nurse cell differentiation. In addition, the results serve to expand the usefulness of oogenesis in Drosophila as a model system for the evaluation of drug-induced metabolic-morphologic abnormalities.

Keratin-like components of gland thread cells modulate the properties of mucus from hagfish (Eptatretus stouti)

SummaryThe hagfishes (cyclostomes) are known to secrete copious amounts of mucus mainly by the holocrine mode from the slime glands. Stressed animals release two types of cells (gland thread cells, GTCs; gland mucous cells, GMCs) which rupture on contact with water and rapidly form a mass of viscous mucus. Herein we report some key sequential events of this process and document a novel role for cytoskeletal polymers. After electrostimulation of Pacific hagfish (Eptatretus stouti), the exudate was collected in a stabilization buffer and GTCs segregated from GMC vesicles. Water was added progressively to mixtures of known quantities of these entities. The changing mucous composition and properties were monitored by light- and electron microscopy, viscometry and immunogold assay. Sequentially, the threads uncoil from GTCs, aggregate with the vesicles, the vesicles rupture and release mucin-like substances, at least some of which adhere to the thread. It was found that the intermediate filament (IF)-rich threads markedly facilitate hydration and modulate the viscoelastic and cohesive properties of the resultant mucus. It was speculated that the thread abets localization of mucus in an aqueous environment and promotes adhesion of mucus to surfaces such as the fish integument. As judged by immunostaining in situ, GTCs, as well as several cell-types in the epidermis, contain keratin-like components. The role of biopolymers on the properties of teleost and mammalian mucus is discussed.

Hagfish Skin and Slime Glands

The scaleless hagfish employs two modes of secretion, holocrine by the slime glands and to a lesser extent merocrine by the epidermis for release of its voluminous viscous exudate. In both cases, large organized entities of intermediate filaments (IFs) are requisite participants. In the epidermis, each small mucous cell contains an IF-rich, basket-like structure (‘capsule’) which serves to compartmentalize the mucin-rich secretory vesicles within the apical region for subsequent release. Each epidermal thread cell contains at least one large IF-biopolymer (‘thread’) which may affect both the physical properties of the epidermis and the epidermal exudate. By contrast, the single IF-rich thread biopolymer precisely localized in each gland thread cell, interacts synergistically with mucins from the gland mucous cells to loosely organize water into a viscous mass of slime (stage 1), which, after physical perturbation, forms even more massive IF-aggregates (‘cables’) accompanied by the release of water (stage 2). The massive gland thread exhibits a linearly aligned IF/microtubule motif and is destined for extracellular export to function in an aqueous environment. Comparisons of the hagfish thread IF-polypeptide sequences (γ and α, with high threonine-contents) reported herein show no preferred identity to any other type of sequenced IF including those from higher vertebrates, a cephalochordate and invertebrates. Inasmuch as several keratin traits exist, we have characterized γ and α as homologues of type I and II epidermal keratins, respectively.

Metabolic-morphologic events in the integument of the Pacific hagfish (Eptatretus stoutii).

SummaryLight- and electron-microscopic autoradiography were used to obtain a coordinated metabolic-morphologic view of some of the events of cellular differentiation that occur across the epidermis of the Pacific hagfish (Eptatretus stoutii) and which enable this animal to secrete copious amounts of mucus. As judged by epidermal incorporation of [3H]-thymidine in vivo, about 98 % of DNA replication is confined to the basal three layers of the total of 6–8 layers of cells. Small mucous cells (SMC), the most numerous of the three major cell types involved in mucigenesis, show in vitro and in vivo radioincorporation profiles of [3H]-L-lysine and [3H]-D-glucosamine which differ markedly from those of [3H]-L-fucose and [3H]-D-galactose. Time-course incorporation profiles (mean silver grains/cell and percentage of cells with at least one cluster of silver grains) of [3H]-L-lysine and [3H]-D-glucosamine not only reflected the metabolic activities of cell renewal and differentiation in basally-located cells but also the high mucigenic activity in cells near the epidermal surface. By contrast, [3H]-L-fucose and [3H]-D-galactose were mainly incorporated by the more mature SMC in juxtanuclear regions near Golgi complexes and newly formed secretory vesicles. The intensity of [3H]-fucose labeling appeared proportional to the intensity of histochemical staining of the apical cytoplasm. The prominent capsule, within SMC in basal and lateral regions, which arises from a tight intermingling of tonofilaments, appears to restrict secretory vesicles to apical regions while the cell progressively differentiates and migrates to the epidermal surface. The other mucigenic cell types, large mucous cells and thread cells, each show distinctive differentiation and radioincorporation patterns.

Structural forms and possible roles of aligned cytoskeletal biopolymers in hagfish (slime eel) mucus

Abstract The hagfish, marine benthic dwellers, are primitive vertebrates which, upon stress, secrete massive quantities of mucus from slime glands of the integument by the holocrine mode. Lysis of each gland thread cell releases a single, intermediate filament-rich thread (∼ 60 cm × 1.5 μm), which collectively modulates the hydration and cohesiveness of mucus. In this report, with the Pacific hagfish (Eptatretus stouti), we showed that the threads exhibit a tendency for self-assembly into novel, aligned and intertwined cytoskeletal aggregates (“cables”). Large cables (15–40 cm in length, 50–800 μm in width) can be physically prepared from the whole mucous exudate, but similar smaller cables (0.1–10 cm in length, 50–200 μm in width) were found occurring naturally, particularly at the ends of laid eggs. Threads and cables may facilitate adhesion of mucus to the integument of the fish, restrict eggs to an optimum spawning site, and abet defense against invasive organisms.

Hemagglutinins in the mucus of Pacific hagfish, Eptatretus stoutii.

Abstract 1. 1. A disc electrophoretic and immunologic comparison was made of Pacific hagfish (Eptatretus stoutii) serum and a concentrated, water-soluble fraction of mucus. 2. 2. Usually 12–15 protein components were obtained by disc electrophoresis of mucus and the profile was similar in many regions to that of serum. 3. 3. Usually at least two and sometimes four proteins of mucus could be shown to be immunologically related by double diffusion. 4. 4. Natural hemagglutinins which were found in mucus by direct hemagglutination were also shown to have heat-sensitivity similar to hemagglutinins in serum.

Autoradiographic studies of protein and polysaccharide synthesis during vitellogenesis in Drosophila

SummaryQuantitative light- and electron-microscopic autoradiography was used to evaluate metabolic processes that occur during late developmental stages (10–14) of oogenesis in Drosophila melanogaster. Major differences in radiolabelling patterns were found after in vivo (10–45 min) uptake of [3H]-monosaccharides and [3H]-L-lysine. Several different methods of data analysis were required to facilitate interpretation of these patterns. [3H]-L-lysine produced extensive cytoplasmic labelling at all developmental stages. In addition, about 15% of alpha yolk spheres were intensely labelled at stage 10, reflecting the incorporation of radiolabelled vitellogenins synthesized during the incubation period. Subsequent stages showed low silver grain density over alpha yolk spheres until stage 14, when a burst of [3H]-L-lysine incorporation by most alpha spheres was observed, possibly indicative of a maturation process for embryogenesis. [3H]-D-glucose and [3H]-D-galactose (10 min, in vivo) both induced intense labelling of the beta yolk spheres in a manner suggesting in situ assembly beginning at early stage 13. Inasmuch as the polysaccharide of beta yolk spheres has the properties of glycogen (e.g., rosette structure digested by α-amylase) and the radiolabelled monosaccharides were introduced intraabdominally, it is evident that transport systems as well as enzymes utilizing glucose and galactose for glycogenesis must be readily available. It is notable that wide-spread labelling of egg chambers was elicited by [3H]-D-glucose and [3H]-D-galactose (e.g., nurse cells, follicle cells, chorion, vitelline membrane), but the labelling induced by [3H]-N-acetylmannosamine was restricted mainly to the endochorion. A possible role of microtubules in distribution and assembly of yolk spheres was inferred when colchicine, admixed to the culture medium (2–5 ppm), produced abnormal distribution and diminution in number of both alpha and beta yolk spheres. In addition to revealing previously unknown metabolic events of vitellogenesis, the results provide additional criteria for stage characterization as well as a means to specifically label certain macromolecules for purposes of isolation.

Metabolic-morphologic characteristics of the integument of teleost fish with mature lymphocystis nodules

SummaryNormal and virus-infected (lymphocystis disease) integument from five species of teleosts was examined by light and TEM autoradiography and SEM to establish metabolic-morphologic characteristics of integument with mature lymphocystis cells (LCs). LCs with numerous morphologic attributes of a late developmental stage showed highest incorporation of [3H]-thymidine in vivo (1–91 h) above the intracytoplasmic inclusion body (ci) with little radiolabel in nuclei, cytoplasmic icosahedral deoxyriboviruses (ICDVs) or capsule. Analysis by quantitative autoradiography revealed that the % total cell label in ci and cytoplasm did not vary appreciably from 1–91 h and was corroborative with morphologic criteria of maturity. A possible phylogenetic difference was noted between teleosts, wherein normal integument showed uptake of [3H]-thymidine in vivo (1 h) by cells at all levels of the epidermis, and cyclostomes (Spitzer et al. 1979) wherein labeling was confined to the basal third of the epidermis. Among four infected teleost species, the mean diameters of the ICDVs measured under the same conditions, ranged from 259.5 nm to 290.0 nm with the mean for each species differing significantly (p < 0.01) from each of the other means. Ruptured LCs were shown by TEM and SEM to have released ICDVs onto the lesions and integument. Various stages of LC degeneration, host response, and integumental repair processes were documented. An evaluation of labeling in vivo of the capsular matrix was compatible ([3H]-D-galactose> [3H]-L-lysine ≫ [3H]-L-fucose) with a glycosaminoglycan-protein structure.

Subcellular Localization of Monoamine Oxidase in Bovine Thalamus Tissue using Immunoferritin Conjugates

A combination of discontinuous sucrose gradient analysis and polyacrylamide electrophoresis was used to isolate one of the multiple forms of monoamine oxidase (MAO) from bovine thalamus. This substance (the principle form and the most anodic of the five MAO forms observed) was used as a basis for an immunoferritin-electron microscope approach to determine the subcellular localization of MAO in thalamus. This form of MAO, as well as antigenically related forms, was found to reside mainly on the outer mitochondrial membrane. In addition, the action of SDS on solubilization and interconversion of MAO forms was studied and found to be dependent on the concentration and time of reaction of SDS with thalamus tissue.