Ultrastructural localization of spicule matrix proteins in normal and metalloproteinase inhibitor-treated sea urchin primary mesenchyme cells

Studies of the sea urchin larval skeleton have contributed greatly to our understanding of the process of biomineralization. In this study we have undertaken an investigation of the morphology of skeleton formation and the localization of proteins involved in the process of spicule formation at the electron microscope level. Sea urchin primary mesenchyme cells undergo a number of morphological changes as they synthesize the larval skeleton. They form a large spicule compartment that surrounds the growing spicule and, as spicule formation comes to an end, the density of the cytoplasm decreases. Inhibition of spicule formation by specific matrix metalloproteinase inhibitors or serum deprivation has some subtle effects on the morphology of cells and causes the accumulation of specific classes of vesicles. We have localized proteins of the organic matrix of the spicule and found that one protein, SM30, is localized to the Golgi apparatus and transport vesicles in the cytoplasm as well as throughout the occluded protein matrix of the spicule itself. This localization suggests that SM30 is an important structural protein in the spicule. Another spicule matrix protein, SM50, has a similar cytoplasmic localization, but in the spicule much of it is localized at the periphery of the spicule compartment, and consequently it may play a role in the assembly of new material onto the growing spicule or in the maintenance of the integrity of the matrix surrounding the spicule.     

Ultrastructural investigation of spicule formation in the gorgonianLeptogorgia virgulata (Lamarck) (Coelenterata: Gorgonacea)

Summary Ultrastructural examination of original and regenerated branch tips of the gorgonianLeptogorgia virgulata reveals that spicule formation begins with the aggregation of scleroblasts in the mesoglea. Calcite crystal deposition occurs within a Golgi vacuole containing organic matrix. Vacuole size increases while matrix incorporation and subsequent crystal growth continue, filling the vacuole. At approximately this time, the scleroblasts dissociate and wart formation begins. Further spicule growth stretches the cell into a thin envelope. Fusion of vacuole and plasma membrane followed by breach formation during spicule growth, as well as scleroblast atrophy or migration from mature spicules, result in the transition of the spicule from the intracellular to the extracellular environment. The results also reveal aborted spicules and digestive bodies, implying possible relationships among calcification, detoxification, and waste management.Contribution No 436, Belle W. Baruch Institute for Marine Biology and Coastal Research, University of South Carolina, Columbia, South Carolina, 29208, USA     

Immunogold detection of glycoprotein antigens in sea urchin embryos

Four developmental stages of sea urchin embryos were labeled with colloidal gold in an attempt to elucidate the intracellular trafficking patterns within the cells that produce the glycoprotein matrix of the embryonic spicule. The primary mesenchyme cells (PMCs) form a syncytium and secrete an organic matrix on which calcium carbonate is laid down to form an endoskeletal spicule. The organic matrix has been isolated and characterized as glycoprotein consisting of four major bands. Polyclonal antibodies to these glycoproteins were used to label embryos from the mesenchyme blastula, early gastrula, late gastrula, and plutei stages of development. The label is concentrated in the Golgi complex and associated vesicles, in secretory vesicles, and in the organic matrix. The density of the labeling increases as development proceeds.     

Cytodifferentiation of the adenohypophysis of the domestic fowl

Summary In the chick embryo the first membrane-bound secretory granules occur in the cytoplasm of occasional cells in the cephalic lobe of pars distalis at the 7th day of incubation. On the 8th day most of the cells in both the cephalic and caudal lobes contain secretory granules that are variable in size, form and density.On the 9th day at least two types of glandular cells are distinguishable in the cephalic and in the caudal lobes; however, these cells are not comparable with those of the adult gland. Differentiation of acidophils and basophils occurs, apparently simultaneously, in 11-day embryos.The cells of the cephalic and caudal lobes are morphologically distinct from their first appearance. Thus it is concluded that these two lobes develop independently and differently from an early stage of ontogenesis.The secretory granules are formed in the Golgi area of the hypophysial cells after the 8th day of incubation. However, secretory material may be synthesized also by a process not involving the Golgi apparatus.Nerve fibers containing granules first appear in the superficial layer of the median eminence on the 8th embryonic day and by the 12th day three types of granules and two types of clear vesicles are identifiable.The investigation reported herein was supported by grant from Japan-U.S. Cooperative Science Program of Japan Association for Science Promotion to Professor Mikami and by U.S.-Japan Cooperative Science Program Grant No. GF-33334 to Professor Farner.     

Morphology and Construction of the Shell Wall in an Agglutinate Soil Testate Amoeba Phryganella acropodia (Rhizopoda)

ABSTRACT. When grown in culture, the soil testate amoeba Phryganella acropodia constructs a shell with or without mineral grains, but of identical morphology. The availability of organic shells, from these cultures, allows for detailed examination of the formation of organic building units in the cytoplasm and their subsequent use as a network in the shell matrix. Organic building units are initially formed as spherical membrane bound vesicles at the margins of dictyosomes, but mature in the cytoplasm by incorporating additional material, some have an electron dense core. These units remain pliable until they are moulded into the shell matrix. Another series of vesicles indistinguishable during initial formation from the organic building units, when mature contain a mass of small elements. This material is discharged at the same time as the building units and is thought to be the binding cement. Inorganic chemical elements appear to be incorporated within the inner lining of the matrix and the alveoli of the organic building units; manganese being preferentially absorbed under culture conditions.     

Ultrastructural Aspects of Spicule Formation in the Solitary Ascidian Herdmania momus (Urochordata,Ascidiacea)

The solitary stolidobranch ascidian Herdmania momus contains numerous calcium carbonate spicules in its tunic and body tissues. The slender body spicules form inside complex sheaths in the body wall and branchial basket, where they remain for the life of the animal. The much smaller tunic spicules form inside the tunic blood vessels and then migrate to the tunic surface, where they become anchored by their spiny base. This paper is an ultrastructural investigation of the formation of the body spicules; the tunic spicules, which apparently form quite differently, will be the focus of a future study. The body spicules are composed of rows of closely packed acicular spines which form completely extracellularly. The spine tips are covered by flattened, highly pseudopodial sclerocytes bound together by tightly interdigitating cell processes. The basal regions of contiguous spines are covered by very thin sclerocyte cell processes. An organic matrix is present within the spines; its exact nature is not clear. A very dense extracellular inter-spine matrix is located between the spine tips and the contiguous basal regions. Presclerocytes within the sheaths between the spicules are probably responsible for formation of the extracellular structures of the sheaths. The presclerocytes appear to aggregate and transform into sclerocytes at the apical end of the spicule. New spines are added at the apical end of the spicule as well as between larger spines. Comparisons are made between body spicule formation in H. momus and skeletogenesis in echinoids.     

A fine structural study of the testicular wall and spermatogenesis in the crinoid,Florometra serratissima (Echinodermata)

The testicular wall and the process of spermatogenesis in the crinoid, Florometra serratissima, has been studied at the fine structural level. The testicular wall is composed of three layers: a perivisceral layer consisting of nerve processes, muscle fibers, and epithelial cells; a haemal sinus containing haemal fluid, collagen-like fibers, and haemocytes; and a germinal layer consisting of germinal and interstitial cells. The germinal layer is elaborated into numerous folds that project into the lumen of the testis and a branch of the haemal channel extends through the core of each fold. Evidence suggesting that nutrients are carried to the testis and germinal cells via the haemal system is presented. Spermatogonia are concentrated around the base of each fold and spermatocytes line the more distal regions. Spermatids occur at the luminal surface of the germinal layer and spermatozoa fill the testicular lumen. Interstitial cells phagocytize spermatozoa and may also transfer nutrients to spermatids. The nucleus of spermatogonia is large and contains one or two nucleoli. The cytoplasm contains numerous organelles, lipid granules, and a distal and proximal centriole, each with a satellite complex. A striated rootlet extends from the distal centriole. During first meiotic prophase, the distal centriole loses its striated rootlet and produces a flagellum, the proximal centriole loses its satellite complex, the nucleolus disappears, and proacrosomal vesicles are synthesized by the Golgi complex. During spermiogenesis, most of the mitochondria appear to fuse to form a single, large mitochondrion, the nuclear chromatin condenses, and superfluous cytoplasm is lost by autophagocytosis. The formation and definitive positioning of the acrosomal vesicle and periacrosomal material at the apex of the nucleus is described in detail.     

Collagen metabolism and spicule formation in sea urchin micromeres

The role of collagen or collagen-like protein(s) in the in vitro formation of the sea urchin embryonic skeleton was investigated using isolated micromeres of Strongylocentrotus purpuratus. Micromeres were cultured in sea water containing 4% horse serum on tissue culture plastic or an extracellular matrix of type I collagen. The effect of proline analogs and an inhibitor of collagen hydroxylation on in vitro spicule formation in both culture systems was monitored. When micromeres are cultured in the presence of proline analogs l-azetidine-2-carboxylic acid and l-3,4-dehydroproline which disrupt collagen metabolism, spicule formation is significantly less inhibited on a collagen substratum than on plastic. Culturing micromeres on plastic in the presence of α,α′-dipyridyl, an inhibitor of collagen hydroxylation, resulted in almost complete inhibition of spicule formation. The inhibition by α,α′-dipyridyl can be overcome by culturing micromeres on collagen substratum. These results do not support the idea of collagen being the calcified organic matrix of the spicule. Rather, they suggest that micromeres synthesize a collagen-like extracellular matrix which is necessary for spicule formation. Inhibition of this activity by proline analogs or a collagen processing inhibitor can be overcome by providing the cells with a previously deposited extracellular matrix.     

Silica deposition in Demosponges: spiculogenesis in Crambe crambe

Transmission electron-microscopy images coupled with dispersive X-ray analysis of the species Crambe crambe have provided information on the process of silica deposition in Demosponges. Sclerocytes (megasclerocytes) lie close to spicules or surround them at different stages of growth by means of long thin enveloping pseudopodia. Axial filaments occur free in the mesohyl, in close contact with sclerocytes, and are triangular in cross section, with an internal silicified core. The unit-type membrane surrounding the growing spicule coalesces with the plasmalemma. The axial filament of a growing spicule and that of a mature spicule contain 50%-70% Si and 30%-40% Si relative to that contained in the spicule wall, respectively. The extracellular space between the sclerocyte and the growing spicule contains 50%-65%. Mitochondria, vesicles and dense inclusions of sclerocytes exhibit less than 10%. The cytoplasm close to the growing spicule and that far from the growing spicule contain up to 50% and less than 10%, respectively. No Si has been detected in other parts of the sponge. The megascleres are formed extracellularly. Once the axial filament is extruded to the mesohyl, silicification is accomplished in an extracellular space formed by the enveloping pseudopodia of the sclerocyte. Si deposition starts at regularly distributed sites along the axial filament; this may be related to the highly hydroxylated zones of the silicatein-alpha protein. Si is concentrated in the cytoplasm of the sclerocyte close to the plasmalemma that surrounds the growing spicules. Orthosilicic acid seems to be pumped, both from the mesohyl to the sclerocyte and from the sclerocyte to the extracellular pocket containing the growing spicule, via the plasmalemma.     

Scleroblast cultures from the gorgonianLeptogorgia virgulata (Lamarck) (Coelenterata: Gorgonacea)

Summary Scleroblasts were separated from fragmented tissue of growing tips ofLeptogorgia virgulata and cultured using a modification of the technique of Rannou. Replacement of fetal bovine serum with horse serum seemed to increase scleroblast viability. Cell adhesion occurred from 14 to 43 d. Cultured scleroblasts demonstrated cell aggregation, spicule formation, and extrusion of spicules into the external medium. Cells showing spicules in the process of being extruded appeared on the average after 24 d of culture. Variability among cultures was marked with respect to both division and spicule formation. Healthy cultures were maintained for more than 4 mo. This work was supported by National Science Foundation grants PCM8201389 and DCB8502698. This is contribution No. 674 of Belle W. Baruch Institute for Marine Biology and Coastal Research, University of South Carolina.     

The cytology of the testaceous rhizopod Lesquereusia spiralis (Ehrenberg) Penard. I. Ultrastructure and shell formation

Ultrastructure and shell formation in the testaceous ameba, Lesquereusia spiralis, were investigated with both scanning and transmission electron microscopy and X-ray microanalysis. The nucleus, surrounded by a fibrous lamina, contains multiple nucleoli. The cytoplasm, containing a well developed granular endoplasmic reticulum, also contains remnants of starch granules in stages of digestion. Spherical aggregates of ribosome-like particles may be seen. Golgi complexes seem to produce both a nonordered fibrous material and an electron dense vesicle. Only the latter appears to bleb off from the Golgi complex. X-ray microanalysis demonstration of silicon in Golgi vesicles and in some dense vesicles suggests that the fibrous component of the cisternae may take up and concentrate silica to form the electron-dense component of the vesicles. Membrane-bound siliceous crystals are often seen adjacent to the Golgi, suggesting either a Golgi origin or platelet formation in vesicles after release from the Golgi complex. Both electron-dense bodies and siliceous platelets are released from the cell by a process similar to apocrine secretion and may be seen outside the cell in route to the shell during shell morphogenesis. Shell development involves fusion of electron-dense bodies to form a matrix, positioning of siliceous platelets in this matrix parallel to the shell surface, and development of a system of matrix chambers. A particulate glycoconjugate is released to the shell surface upon rupture of the matrix chamber.     

Nuclear maturation of ovine oocytes in cultured preantral follicles

Small (150–250 μm in diameter) and large (251–400 μm in diameter) preantral follicles (PFs) in sheep were cultured for 6 days in four different concentrations of transforming growth factor-alpha (TGF-), epidermal growth factor (EGF), FSH and LH. Proportions of follicles exhibiting growth, antrum formation and increase in follicular and oocyte diameter were the initial indicators of development. The ability of the oocytes isolated from these cultured follicles to mature to metaphase II (MII), after 24 h culture in a known in vitro maturation medium was the final criterion of success. TGF- 2.5 ng ml⁻¹, EGF 50 ng ml⁻¹ and FSH 1 and 2 μg ml⁻¹ supported good initial growth of the PFs. Thirty and seventeen percent of the oocytes from the large PFs cultured in TGF- 2.5 ng ml⁻¹ and FSH 2 μg ml⁻¹ respectively, matured to the MII stage. These proportions for oocytes from small PFs were 11 and 6%, respectively. Oocytes from follicles cultured in EGF did not mature to the MII stage. LH at all concentrations tested and TGF-, EGF and FSH above 5, 50 ng ml⁻¹ and 2 μg ml⁻¹, respectively, induced degeneration of the PFs. It was concluded that (i) TGF- 2.5 ng ml⁻¹ supports development of large PFs in sheep to obtain meiotically competent oocytes, (ii) PFs > 250 μm in initial diameter develop better in vitro, and (iii) in vitro development of sheep PFs could be obtained independent of gonadotropin stimulation.     

Transmission microscopy of freeze dried,unstained epiphyseal cartilage of the guinea pig

Summary The question of the initial mineralization in the epiphyseal plate has been investigated to date in specimens prepared by conventional electron microscopical techniques. As conventional techniques can produce artifacts, either a loss of mineral substance or a secondary nucleation, the mineralization process was investigated using freeze dried, vacuum embedded growth cartilage which was neither contrasted nor stained and which had a very short contact with water.The prevailing theory that the first mineralization begins within extracellular matrix vesicles and that the mineralization outside these vesicles is a secondary process was confirmed. Mineralized matrix vesicles were found in the fully mineralized long septa down to the opening zone. In several cases a mineralization could be observed in those transverse septa in which organic substance was present between the cells. The typical radial arrangement of the apatitic needles and platelets in the matrix vesicles could be explained by the formation of a mineralization in an ionotropic gel, the orientation of the matrix macromolecules to be produced by a vectorial influx of calcium ions and phosphate groups coming from different directions. Thin strands of mineral substance with low contrast, which follow the direction of the longitudinal septum, were assumed to be the mineralized collagen fibrils. In several needles dot-like formations were seen and the distance between the middle of neighbouring dots was found to lie mainly in the range 30–56 Å, while the lateral separation distance between the middle of closely packed parallel chains and needles was found to lie mainly in the range 30-42 Å. Parallel periodic structures which could be visualized in apatitic chains and needles 20–40 Å in diameter were assumed to be the 8.2 Å-(100)-lattice planes of apatite, being an indication that these formations already possess criteria of the apatite lattice.We express our thanks to the Deutsche Forschungsgemeinschaft for financial support and to Dr. A. Boyde, London, for valuable discussions.     

Cytochemical reaction for cationic proteins as a marker of primary granules during development in chick heterophilis.

The ammoniacal silver reaction (ASR) for cationic proteins was used as a cytochemical marker for the primary or A granules in the cytoplasm of developing heterophils of chick bone marrow. The presence of the electron-dense particulate reaction product of silver, which is localized in the fully formed rod-shaped A granules, provides a marker by which the A granules could be distinguished from the B granules of similar size and by which the formation and maturation of both granule types could be followed through the developmental stages. Progressive developmental stages were ascertained on the basis of decreasing cell size, increasing condensation and margination of the chromatin, and the number and morphology of the granules; the stages were divided into promyelocyte, myelocyte, metamyelocyte and heterophil. During the promyelocyte stage, the first appearance of the electron-dense, membrane-bound, spherical granules (0.3--1.0 micrometer in diameter) is observed in the vicinity of an extensive Golgi complex. They occur in a cytoplasm containing rough-surfaced endoplasmic reticulum, ribosomal clusters, centrioles, mitochondria, microtubules, as well as the membranes, saccules, vesicles and vacuoles of the Golgi complex. These granules are considered as primary but their presence as the only granule type appears very brief. The ASR reaction product is first detected on the surface of these primary granules in late promyelocytes or myelocytes. The secondary or B granule, devoid of reaction for cationic protein at all stages, appears as a condensing vacuole in promyelocytes, but after some A granules are already present. The vacuole contents condense to form the B granules which are 0.1--0.6 micrometer in diameter, often oval-shaped, and contain a loose filamentous material surrounded by a membrane. Tertiary C granules or lysosomes appear during the myelocyte stage as dense core vesicles (0.1--0.2 micrometer in diameter) negative for cationic protein.     

Development of the axon cap neuropil of the Mauthner cell in the goldfish

Summary Development of the axon cap neuropil of the Mauthner neuron in post-hatching larval goldfish brains was observed electron-microscopically. The axonal initial segment of newly hatched (day-4) larvae is completely covered with synaptic terminals containing clear spherical synaptic vesicles. Profiles of thin terminal axons, the spiral fibers, containing similar synaptic vesicles, rapidly increase in number around the initial segment and form glomerular neuropil similar to the central core of the adult axon cap by day 7. Three types of synapses are formed in the core neuropil. Bouton-type synapses contacting the initial segment are most abundant in day-4 to-14 larvae; they decrease thereafter and are rare on the distal half of the initial segment of day-40 larvae. Asymmetric axo-axonic synapses are commonly observed between spiral fibers in the core neuropil of day-7 to -19 larvae, but become fewer by day 40. Unique symmetrical axo-axonic synapses showing accumulation of synaptic vesicles on either side of apposed membrane thickenings first appear in day-14 core neuropil, gradually increase in number, and become the predominant type in day-40 core neuropil. Thick myelinated axons, which lose their myelin sheaths in the glial cap cell layer, start to penetrate into the axon cap on day 10. They gradually increase in number and form the peripheral part of the axon cap together with the cap dendrites, which finally grow into the axon cap from the axon hillock region of the Mauthner cell by day 40.     

荞麦糊粉层和子叶中贮藏蛋白质积累过程的超微结构

应用透射电镜技术对荞麦（Fagopyrum esculentum)子叶和糊粉层细胞中贮藏蛋白质的积累过程进行了研究。荞麦开花后15天,胚乳最外细胞的液泡中开始积累蛋白质。开花后25天,最外层胚乳细胞中积累较多的糊粉粒（直径1－2μm)形成糊粉层。开花后20天,子叶细胞中蛋白质开始在液泡和细胞质中积累,同时液泡通过膜的向内生长和缢裂两种方式形成体积较小的液泡。开花后25天,成熟的子叶细胞中含有丰富的蛋白质,贮藏蛋白质主要积累在液泡中形成体积较大的蛋白质贮藏液泡（PSVs,protein storage vacuoles,直径1－3μm）。在荞麦子叶积累蛋白质的各个阶段,细胞质中都有一些来源于高尔基体,含蛋白质的电子不透明小泡（直径0.1-0.7μm)存在,观察到有些小泡正进入液泡,推断这种来自高尔基体膜囊的小泡不仅将蛋白质运输到液泡形成PSVs的作用,也可能是荞麦成熟子叶积累贮藏蛋白质的一种结构。     

Ultrastructure of collagen in sea urchin embryos

Summary Collagen fibrils with a main period banding of 610 Å and 220 Å in width were observed in the blastocoel of 72-h embryos of the sea urchin,Strongylocentrotus purpuratus. Non-striated fibrils of 50 Å diameter were also observed. The collagen is seen in highest concentration in the vicinity of mesenchyme cells which are richly endowed with endoplasmic reticulum and secretory vesicles. A role for collagen in cell attachment, orientation and spicule formation is discussed.     

Opioid modulation of capsaicin-evoked release of substance P from rat spinal cord in vivo

Capsaicin has been shown to evoke the release of substance P (SP) from small diameter primary afferent fibers. Using an in vivo perfusion of the rat spinal cord, this study examined the pharmacology of opioid receptor systems which modulate the capsaicin-evoked release of SP. The addition of capsaicin (200 μM) to the perfusate raised SP-like immunoreactivity (SP-LI) from resting levels of 31±5 to 74±14 pg/ml or an increase of 139% above the baseline. Using high pressure liquid chromatography (HPLC) the identity of the released SP-LI was determined to coelute primarily with authentic SP or the oxidized form of SP. Opioid receptor agonists were added to the perfusate and their ability to inhibit capsaicin-evoked release of SP-LI was assessed. Morphine (10–100 μM), DAGO (1–100 μM), DPLPE (10–100 μM), but not U50488H (100 μM) produced a dose-dependent reduction in the capsaicin-evoked release of SP-LI. Pretreatment with the opioid receptor antagonist naloxone (1 mg/kg, IP) had no effect on the basal or capsaicin-evoked release of SP-LI. Naloxone pretreatment was able to antagonize completely the opioid-produced inhibition of capsaicin-evoked SP-LI release. These data indicate that the release of SP from primary afferent fibers can be modulated by the activation of mu or delta but not kappa opioid receptors. Further, these data support the hypothesis that spinally administered mu and delta opioid agonists may produce their antinociceptive effect through the presynaptic inhibition of neuropeptide release from small diameter primary afferent fibers.     

Studies on the Cellular Pathway Involved in Assembly of the Embryonic Sea Urchin Spicule

Micromeres from the 16-cell stage sea urchin embryo were isolated and cultured in vitro in seawater containing 3% horse serum. Under these conditions these cells differentiate into spicule-forming, primary mesenchyme cells. To obtain insight into the route traveled by Ca²⁺ to form the pseudocrystalline spicule composed of CaCO₃ and matrix proteins, studies with various inhibitors were undertaken. Experiments with members of several different classes of Ca²⁺ channel blockers established that the Ca²⁺ utilized for spiculogenesis must be taken up by the cells. Moreover, studies using two agents that disrupt the endomembrane system, monensin and brefeldin A, showed that both blocked spicule formation. Based on these experiments, we conclude that extracellular Ca²⁺ must enter the primary mesenchyme cells prior to being deposited extracellularly as CaCO₃ and that this ion and/or the matrix proteins found in the spicule are routed through the secretory pathway that has been established to exist in a wide variety of other cell types.     

ULTRASTRUCTURAL STUDY ON THE SCLEROBLAST OF ORYZIAS LATIPES DURING ETHISTERONE-INDUCED ANAL-FIN PROCESS FORMATION

Cytodifferentiation of the scleroblast during ethisterone-induced anal-fin process formation in adult females of Oryzias latipes was observed electron-microscopically. Scleroblasts seem to arise from immature mesenchymal cells located not only along the postero-external margin of the joint plates, but also in the space between paired joint plates. These cells contained a small nucleus with condensed chromatin and a very small amount of cytoplasm with a marked paucity of formed organelles. These cells proliferated and/or migrated to the process-forming site of the joint plates and aggregated to the mass. Young scleroblasts were spindle-shaped in profile and contained an enlarged nucleus with dispersed chromatin. Rough-surfaced endoplasmic reticulum was developed in the cytoplasm. Scleroblasts became oval in shape and commenced to secrete bone matrix. Well-developed scleroblasts were characterized by an extensive development of rough-surfaced endoplasmic reticulum. This seems to correlate with the active protein synthesis required by the scleroblast for laying down bone matrix. As the young horny process developed, scleroblasts covered its apex, and became flattened. Some scleroblasts which became enclosed in the developing matrix had much the same characteristics as when they were lying on its surface.