Adipocyte morphology during hormone-induced lipid deposition and mobilization: An ultrastructural investigation in the perfused cardiac fat

The rat pericoronary adipose tissue was perfused in the presence of either the liposynthetic hormone insulin or the lipolytic hormone noradrenaline. Insulin perfusion associated with a) larger adipocyte mean sectional diameter in comparison with noradrenaline perfusion; b) glycogen deposition; c) appearance of small fat globules at discrete sites at the periphery of the main lipid drop. The two latter phenomena were apparently dose-dependent. Massive lipid deposition was induced by addition of triglycerides to the perfusion medium and this associated with appearance of prominent endoplasmic reticulum in the cytoplasm. In noradrenaline-perfused adipose tissue many small lipid droplets surrounded the central lipid deposit and the endoplasmic reticulum was in the form of both thin long, dashed cisternae sometime surrounding lipid droplets and grouped, anastomosing tubular cisternae. The present work shows that the perfused white adipose tissue of the heart is a suitable model to study, in situ, the morphological effects of hormones in adipocytes.     

DIFFERENTIATION OF INTRACAPSULAR CELLS IN THE SPOROPHYTE OF SPHAEROCARPOS DONNELLII

Ultrastructural and histochemical changes during intracapsular cell differentiation in the premeiotic sporophyte of the liverwort Sphaerocarpos donnellii Austin were studied. From an initially undifferentiated meristematic tissue, spore mother cells and nutritive cells become differentiated. The first indications of ultrastructural differentiation into two cell types are the accumulation of lipid within spherosomes and the occurrence of plastid tubules in the presumptive spore mother cells. Once differentiated the two cell types are clearly distinguishable on the basis of cytoplasmic vacuolation, stored food reserve, and cell and nuclear size. The mature spore mother cell contains many spherosomes, small vacuoles, starch-containing plastids, and a large central nucleus. The mature nutritive cell, on the other hand, is extremely vacuolate and contains large, starch-filled plastids, a few spherosomes, and a small nucleus. A previously undescribed type of cell was observed in developing sporophyte capsules. This cell is located peripherally in the capsule and degenerates during differentiation of spore mother cells and nutritive cells.     

Increased sterol formation insaccharomyces cerevisiae. Analysis of cell components and ultrastructure of vacuoles

In Saccharomyces cerevisiae nitrogen limitation under aerobic conditions (low specific growth rate) provokes an enhanced synthesis of sterols. Analysis of east cultures during the enhanced sterol biosynthesis showed a temporary decrease of protein content and a simultaneous increase in polysaccharide and lipid levels. This was reflected in the ultrastructure of cells where numerous lipid globules (spherosomes, oleosomes) appeared around extensive membrane-bound compartments containing membrane vesicles and lipoprotein material. Electronograms showed that such compartments were formed between the layers of endoplasmic reticulum and belonged to the vacuome phase of the yeast cell. It appears that vacuoles formed in yeast during enhanced synthesis of sterols have a storage rather than a lysosomal function.     

Gravisusception by buoyancy: a mechanism ubiquitous among fungi?

Gravitropism is ubiquitous among the fungal taxa; however, the mechanism(s) of gravisusception have overall remained obscure so far. In the vegetative sporangiophore of the zygomycete Phycomyces blakesleeanus some 200 large lipid globules form a conspicuous spherical complex which is positioned in a dense mesh of filamentous actin about 100 microm below the growing tip of the apex. Experimental suppression of that complex by transient growth at low temperature greatly diminishes the gravitropic response of the sporangiophore. With respect to size and abundance of the globules, the complex of lipid globules meets basic physical criteria for a possible function of gravisusception. Accumulations of similar lipid globules of critical size are documented in the apex of gravitropically growing hyphae of the endomycorrhizal fungus Gigaspora margarita (Glomeromycota) and have been described in the hyphal apices of members of various fungal phyla. We suppose that--in contrast to plants which use starch as a carbon storage and amyloplasts as statoliths--the fungi utilise the buoyancy of carbon-storing oil droplets for gravisusception.     

SPHEROSOMES AND MITOCHONDRIA IN THE LIVING FUNGAL CELL

Study of living hyphae of Fusarium oxysporum Schlect., Fomes annosus (Fries) Cooke, Ceratocystis fagacearum (Bretz) Hunt, Basidiobolus ranarum Eidam, and Mycotypha microspora Fenner with phase contrast revealed that these fungi have spherosomes similar to those in vascular plants. The spherosomes are conspicuous in the hyphal tip, suggesting some function other than fat synthesis. It may be that the Woronin bodies reported by other workers are spherosomes. Mitochondria in these fungi are highly pleomorphic and exhibit saltatory movement. They often interact with nuclei in a manner suggesting close membrane contact.     

Studies on the cytophysiology of the fat body of the American silkmoth

Summary A developmental study at the electron microscopic level was conducted of the fat body cells of Hyalophora cecropia (L.). During the last larval instar the fat body increases in volume and the cells exhibit a well developed rough endoplasmic reticulum and protein bodies of diverse sizes. In the pupal fat body, the protein bodies appear to be enclosed by a double membrane and contain glycogen granules, ribosomes and mitochondrion-like structures. In addition, there are large lipid globules, cytolysomes and rough endoplasmic reticulum. The ultrastructure of the protein bodies suggests the development of large bodies by fusion of smaller protein bodies. Changes in fat body cell ultrastructure were followed during adult development and cytological evidence was obtained for the depletion of protein, glycogen and lipid in the female during this period. The female adult fat body cell contains free ribosomes, protein bodies, many mitochondria, a few lipid globules and glycogen granules. The male moth fat body cells have many mitochondria, a few glycogen granules, essentially no protein bodies, but an abundance of large lipid globules.Studies on the influence of egg maturation on the morphology of the fat body of Hyalophora gloveri (L.) revealed that ovariectomy of pupae yielded adults having more fat body than normal females, and that the fat body cells of the ovariectomized animals contained more glycogen, lipid and protein. Male pupae receiving ovarian implants developed into adults containing eggs and possessed more fat body than normal females but less than normal males. Very few glycogen granules were found in the fat body cells of normal males or males with implanted ovaries.Supported by grant AM-02818 from the National Institutes of Health.We thank Dr. James Oschman for his helpful suggestions and constructive criticisms.     

SEXUAL REPRODUCTION IN THE TOXIC DINOFLAGELLATE GONYAULAX MONILATA1

The sexual cycle of Gonyaulax monilata Howell was observed in stationary cultures and in nitrogen-deficient medium. The armored, isogamous gametes fuse in a characteristic manner with cingula at oblique angles. Nuclear fusion lags slightly behind cytoplasmic fusion. The zygote enlarges for several days. The dark, double-flagellated planozygote encysts within 1–3 wk. Early hypnozygotes are round to ovoid and contain lipid and one or two large golden-yellow globules. As the hypnozygote matures, the globules become smaller and the cytoplasm darkens and pulls from the wall. All cysts examined contained only one nucleus. A very dark, uninucleate post-hypnozygotic cell escapes through an archeopyle and within 24 h divides into daughter cells which divide in 24–48 h forming a small chain. The production of thick walled zygates in culture implies that such resting stages in marine sediments could serve as a source stock for blooms. This species causes toxic red tides and the existence of benthic “seed beds” consisting of hypnozygotes is now plausible.     

NEUTRAL LIPIDS AS MAJOR STORAGE PRODUCTS IN ZOOSPORES OF THE GIANT KELP MACROCYSTIS PYRIFERA (PHAEOPHYCEAE)1

Neutral lipids, consisting primarily of triacylglycerols, were found to be a major form of carbon reserve in zoospores of the giant kelp Macrocystis pyrifera (L.) C. Ag. The fluorescent stain Nile Red revealed large lipid droplets in the posterior end of the cell, which comprised 20–41% of cellular carbon in newly released spores. Flow cytometric analyses of newly released spores stained with Nile Red revealed considerable variation in the neutral lipid content among spores that was independent of spore size. Lipid droplets were consumed during germination in spores maintained either under constant light or in continual darkness. The availability of light appeared to delay, but did not preclude, lipid use. The rate of lipid use during germination varied considerably among germlings with some cells consuming all of their lipid reserves within 5 h after release. In addition to zoospores, lipid droplets were observed in both male and female gametes. Numerous droplets were observed in eggs, while single lipid droplets were observed in sperm. Neutral lipid droplets were not observed in gametophytes or sporophytes except in developing gametes and spores. Large lipid reserves thus seem to be confined to the microscopic life history stages that presumably have relatively high energy demands. By serving as a supplemental fuel reserve, neutral lipids may be important in extending the effective range of zoospore dispersal.     

Lipid inclusions in the vacuoles of Saccharomyces cerevisiae

Summary Spherical or elongated phospholipid inclusions have been observed in vacuoles of Saccharomyces cerevisiae. The periodicity of these lipid inclusions is 56 Å and the material of which they are composed appears to be derived from spherosomes. The spherosomes arise in the cytoplasm, penetrate the tonoplast, and discharge their contents into the vacuole. It is postulated that the phospholipid matrix of the spherosome then aggregates to produce the myelin-like inclusions. Tween 80 and ergosterol together in the growth medium do not prevent the formation of the lipid inclusions.     

Intracellular origin and secretion of milk fat globules

The cream or fat fraction of milk consists of fat droplets composed primarily of triacylglycerols that are surrounded by cellular membranes. In this review we discuss what is known about how these droplets are formed in and secreted by mammary epithelial cells during lactation. This secretion mechanism, which appears to be unique, is unlike the exocytotic mechanism used by other cell types to secrete lipids. Milk fat globules originate as small, triacylglycerol-rich, droplets that are formed on or in endoplasmic reticulum membranes. These droplets are released from endoplasmic reticulum into the cytosol as microlipid droplets coated by proteins and polar lipids. Microlipid droplets can fuse with each other to form larger cytoplasmic lipid droplets. Droplets of all sizes appear to be unidirectionally transported to apical cell regions by as yet unknown mechanisms that may involve cytoskeletal elements. These lipid droplets appear to be secreted from the cell in which they were formed by being progressively enveloped in differentiated regions of apical plasma membrane. While plasma membrane envelopment appears to be the primary mechanism by which lipid droplets are released from the cell, a mechanism involving exocytosis of lipid droplets from cytoplasmic vacuoles also has been described. As discussed herein, while we have a general overview of the steps leading to the fat globules of milk, virtually nothing is known about the molecular mechanisms involved in milk fat globule formation, intracellular transit, and secretion.     

The supression of milk fat globule secretion by colchicine: an effect coupled to inhibition of exocytosis

The effects of colchicine on release of milk lipids from mammary tissue were evaluated by biochemical analysis of milk and morphological study of the tissue following intramammary infusions of the alkaloid into lactating goats. Colchicine produces a reversible drop in milk yield. As the flow of milk resumes, 36–48 h after infusion, the fat content of the milk increases, phospholipid per g of total globule lipid falls, mean size of milk fat globules increased and diameters of fat droplets (presecretory milk fat globules) within lactating cells approximately double. These observations are consistent with the conclusion that colchicine suppresses milk fat globule secretion but that globules continue to grow in size within cells during the suppression period. These findings indicate that secretion of milk fat globules and the skim milk phase are coupled.     

Ultrastructure of the milk fat globule membrane with and without triglyceride

Summary The primary milk fat globule membrane (MFGM) around freshly secreted milk fat globules consists of a unit membrane separated from the triglyceride core by a dense material. This dense material may widen to include cytoplasmic organelles or may form small blebs. Preincubation and fixation of the globules at temperatures between 4° C and 60° C has no effect on the width or appearance of the dense material. Isolated MFGM profiles show structures identical to those found on intact globules. The dense material on the isolated MFGM profiles is unaffected by extractions which remove essentially all the triglyceride present in the pellets of MFGM.The structure of the primary MFGM is therefore independent of any triglyceride content and the earlier suggestions that the dark material represented a triglyceride layer of high melting point adsorped during cooling of the globules after milking are not supported by the work described in this paper.     

Regulation of Lipid Droplet Size in Mammary Epithelial Cells by Remodeling of Membrane Lipid Composition—A Potential Mechanism

Milk fat globule size is determined by the size of its precursors—intracellular lipid droplets—and is tightly associated with its composition. We examined the relationship between phospholipid composition of mammary epithelial cells and the size of both intracellular and secreted milk fat globules. Primary culture of mammary epithelial cells was cultured in medium without free fatty acids (control) or with 0.1 mM free capric, palmitic or oleic acid for 24 h. The amount and composition of the cellular lipids and the size of the lipid droplets were determined in the cells and medium. Mitochondrial quantity and expression levels of genes associated with mitochondrial biogenesis and polar lipid composition were determined. Cells cultured with oleic and palmitic acids contained similar quantities of triglycerides, 3.1- and 3.8-fold higher than in controls, respectively (P < 0.0001). When cultured with oleic acid, 22% of the cells contained large lipid droplets (>3 μm) and phosphatidylethanolamine concentration was higher by 23 and 63% compared with that in the control and palmitic acid treatments, respectively (P < 0.0001). In the presence of palmitic acid, only 4% of the cells contained large lipid droplets and the membrane phosphatidylcholine concentration was 22% and 16% higher than that in the control and oleic acid treatments, respectively (P < 0.0001). In the oleic acid treatment, approximately 40% of the lipid droplets were larger than 5 μm whereas in that of the palmitic acid treatment, only 16% of the droplets were in this size range. Triglyceride secretion in the oleic acid treatment was 2- and 12-fold higher compared with that in the palmitic acid and control treatments, respectively. Results imply that membrane composition of bovine mammary epithelial cells plays a role in controlling intracellular and secreted lipid droplets size, and that this process is not associated with cellular triglyceride content.     

Membranous appendices of spherosomes (oleosomes)

Spherosomes (oleosomes) of cotyledons of rape (Brassica napus L.), sunflower (Helianthus annuus L.), and watermelon (Citrullus vulgaris, Schrad.) seedlings are delimited by a half unit membrane that appears to be continuous with each of the osmiophilic layers of a tripartite unit membrane forming a handlelike appendix of the spherosomes. Prior to any noticeable utilization of the spherosomal storage fat, ribosomes were found to be attached to these handles. At later stages appendices of the spherosomes are smooth, showing a diameter of about 22 nm that greatly exceeds the thickness of any other unit membrane profiles identical in structure and diameter osomes appears to be continuous with the thick lipid layer of the handles. In intermediate stages of fat depletion the spherosomal bodies become invaginated with cytoplasmic material. Finally vesicles with cytoplasmic contents surrounded by a membrane with a typically thick lipid layer are left in the cells. Membrane profiles indentical in structure and diameter to the spherosomal appendices were also present in electron micrographs of the lipolytic membrane fraction recovered from sucrose density gradients after centrifugation of a microsomal cell fraction. The ultrastructural observations are taken for evidence that the spherosomal appendices represent the lipase-carrying membranes isolated previously (Theimer and Rosnitschek, 1978). A novel hypothesis for development and utilization of fat-storing spherosomes is also proposed.     

Ultrastructural and cytochemical aspects of the female gonad of Geoplana burmeisteri (Platyhelminthes, Tricladida, Terricola)

The ultrastructure of the female gonad of the land planarian Geoplana burmeisteri was investigated by means of electron microscopy and cytochemical techniques. It consists of two small germaria located ventral to the intestine and of two irregular, lateral rows of vitelline follicles, both enveloped by a tunica composed of an extracellular lamina and an inner sheath of accessory cells. Accessory cell projections completely surround developing oocytes and vitellocytes. The main feature of oocyte maturation is the appearance of chromatoid bodies and the development of the rough endoplasmic reticulum (RER) and Golgi complexes. These organelles appear to be correlated with the production of egg inclusions of medium electron density, about 1.5-1.8 microm in diameter, which remain scattered in the ooplasm of mature oocytes. On the basis of cytochemical tests demonstrating their glycoprotein composition, these inclusions were interpreted as residual yolk globules. Vitellocytes are typical secretory cells with well-developed RER and Golgi complexes that are mainly involved in the production of yolk globules and eggshell globules, respectively. Eggshell globules appear to arise from repeated coalescence of small Golgi-derived vesicles and, at an intermediate stage of maturation, show a multigranular pattern. Later, after vesicle fusion, they reach a diameter of 1.3-1.6 microm when completely mature and show a meandering/concentric pattern, as is typical of the situation seen in most Proseriata and Tricladida. The content of yolk globules is completely digested by pronase, while the content of eggshell globules is unaffected. Mature vitellocytes contain, in addition, a large quantity of glycogen and lipid droplets as further reserve material. On the basis of the ultrastructural characteristics of the female gonad described above and in relation to the current literature, we conclude that G. burmeisteri appears to be more closely related to the freshwater triclads, in particular to members of the Dugesiidae, than to the marine triclads.     

Microbiological Synthesis of Fat: THE INFLUENCE OF PHOSPHATE CONCENTRATION ON THE METABOLISM OF PENICILLIUM LILACINUM THOM IN SURFACE CULTURE ON A SUCROSE MEDIUM

The influence of phosphate concentration in the medium on therelative amounts of fat, protein, and carbohydrate synthesizedby Penicillium lilacinum Thom has been studied. In a sucrose salts medium in which the C:N ratio was high (65:1)and conducive to fat synthesis, the carbohydrate content ofthe felt was decreased and the fat content increased when theconcentration of sodium dihydrogen phosphate in the medium wasraised from 0.73 to 1.46 per cent. w/v. In a similar mediumin which the C:N ratio was low (65:2) this effect was not observed.In the medium of high C:N ratio with phosphate at the higherlevel, fat accumulated over a longer period than in any othercase. Felts grown with the high C:N ratio and with phosphate at thehigher concentration were richer in protein and in total nitrogenthan were felts developed with the high C:N ratio and less phosphate.This effect was still more pronounced in the medium of low C:Nratio. Change in the initial concentration of nitrate in themedium exercised more marked effects on the metabolism of themould than did change in the initial phosphate level.     

Ultrastructure of Saccharomyces cerevisiae strain AG1-7 and its responses to changes in environment

Asynchronous populations of the budding yeast Saccharomyces cerevisiae strain AG1-7 were examined by freeze-fracture electron microscopy for ultrastructural changes occurring in response to changes in the environment, specifically the following: temperature (23 or 37 degrees C); cell density (exponential, early stationary, and stationary phases); various periods of nitrogen starvation at low cell density, and return of nitrogen-starved cells to nitrogen-replete medium. This information has been gathered in preparation for ultrastructural examination of comparable responses of temperature-sensitive cell-cycle mutants. The plasma membrane was found to be particularly responsive to changes in environment. A high proportion (75%) of cells in exponential phase populations at 37 degrees C displayed paracrystalline arrays of plasma membrane particles, whereas this proportion was much lower (20%) at 23 degrees C in the same medium; plasma membrane grooves were longer at 37 than at 23 degrees C. In budded cells, the mother cell displayed paracrystalline arrays more frequently than the bud. Entry of cells into stationary phase, either through permitting population growth or by limiting nitrogen supply, resulted in increases in numbers of paracrystalline arrays and grooves. Groove depth also increased. The paracrystalline-array and groove-density responses were independent, both during entry into stationary phase and during the subsequent lag phase. Unusual groove forms appeared during stationary phase in high cell density populations, but not in low cell density nitrogen-starved populations. "Aggregate" and "geometric" tonoplast forms, previously described in strain A364A when grown under some of the conditions used here, were not found in AG1-7 under any of the conditions used here. It was demonstrated that particle-free patches can arise rapidly on the tonoplast of AG1-7 in response to temperature change from 37 to 23 degrees C. During stationary phase, spherosomes (lipid droplets) increased in size, particularly in response to nitrogen depletion. After 72 h of nitrogen starvation, about 10% of cell volume consisted of spherosomes. Changes in vacuolar content and mitochondrial form were also noted during entry into stationary phase.     

Calcium carbonate deposition in a cell wall sac formed in mulberry idioblasts

Summary. Although calcium carbonate is known to be a common biomineral in plants, very little attention has been given to the biological control of calcium carbonate deposition. In mulberry leaves, a subcellular structure is involved in mineral deposition and is described here by a variety of cytological techniques. Calcium carbonate was deposited in large, rounded idioblast cells located in the upper epidermal layer of mulberry leaves. Next to the outmost region (“cap”) of young idioblasts, we found that the inner cell wall layer expanded to form a peculiar outgrowth, named cell wall sac in this report. This sac grew and eventually occupied the entire apoplastic space of the idioblast. Inside the mature cell wall sac, various cellulosic membranes developed and became the major site of Ca carbonate deposition. Concentrated Ca²⁺ was pooled in the peripheral zone, where small Ca carbonate globules were present in large numbers. Large globules were tightly packed among multiple membranes in the central zone, especially in compartments formed by cellulosic membranes and in their neighboring membranes. The maximum Ca sink capacity of a single cell wall sac was quantified using enzymatically isolated idioblasts as approximately 48 ng. The newly formed outgrowth in idioblasts is not a pure calcareous body but a complex cell wall structure filled with substantial amounts of Ca carbonate crystals. Correspondence and reprints: Graduate School of Science and Technology, Kyoto Institute of Technology, Goshokaido, Matsugasaki, Sakyo, Kyoto 606-8585, Japan.     

Chemical composition of the membranes of fat globules of cow's milk

The methods of microthin-layer chromatography on plates with silica gel and disc-electrophoresis in PAAG are used to determine the lipid and protein composition of fat globule membranes of cow milk on the first (foremilk) and the tenth day (milk) of lactation as well as of the buttermilk, a by-product of the technological processing of milk. Differences are found in the quantitative content of the basic classes of lipids and proteins in membranes of fat globules produced from foremilk and milk of cows. The membranes of buttermilk and milk fat globules are characterized by the identical qualitative and similar quantitative chemical composition, that permits using buttermilk as easily available and rich source of components from membranes of cow milk fat globules in the first place of phospholipids and sterols.     

Isolation and characterization of peanut spherosomes

Spherosomes of cotyledons of germinating peanuts (Arachis hypogea L.) were examined by electron microscopy and found to be particles about 1.0 to 2.0 μ in diameter bounded by a limiting membrane. Isolated spherosomes appear similar to spherosomes in situ. The isolated spherosomes are composed of 98.1% total lipids, 0.77% phospholipid and 1.27% protein by dry weight. The amounts of protein and phospholipid associated with the isolated spherosomes are sufficient to account for limiting membranes. Spherosomes amply account for the lipid in a peanut cotyledon. The activity of lipase and fatty acyl-Coenzyme A synthetase is not associated with the isolated spherosomes. This suggests that peanut spherosomes are principal sites of lipid storage but not of lipid degradation.