IDENTIFICATION OF GLYCOGEN IN THIN SECTIONS OF AMPHIBIAN EMBRYOS

Transcription

1 J. Cell Sci. a, (1967) 257 Printed in Great Britain IDENTIFICATION OF GLYCOGEN IN THIN SECTIONS OF AMPHIBIAN EMBRYOS MARGARET M. PERRY Institute of Animal Genetics, Edinburgh SUMMARY Embryonic amphibian cells when examined with the electron microscope were observed to contain an abundance of small particles, approximately 325 A in diameter. The periodic acid/ Schiff reaction and enzymic digestion were employed to determine the nature of the particles, and from the results of these tests they were concluded to be glycogen. Treatment of thin sections with periodic acid/lead citrate solutions resulted in a marked increase in contrast of the glycogen particles compared with other cell structures, and in a clearly defined substructure of 40-A grains appearing within the particles. This differential staining method enabled the particulate glycogen to be distinguished from ribosomes. INTRODUCTION The presence of glycogen in amphibian eggs and embryos and its utilization during development have been well established (Jaeger, 1945; Gregg, 1948). At the ultrastructural level, however, little mention has been made of this important storage material which, in the axolotl for example, accounts for as much as 16-5% of the dry weight of the dorsal region of the gastrula (Heatley & Lindahl, 1937). In a study of the developing anuran 9ucker, Eakin (1964) has tentatively identified particulate structures as glycogen. On the other hand there are many reports, summarized by Revel (1964), on the appearance of glycogen in electron-microscope studies of other organisms. During recent investigations into the fine structure of amphibian embryos it was observed that with the use of the double fixation method of Sabatini, Bensch & Barrnett (1963), the bulk of the cytoplasm contained a particulate component which had not been so apparent in osmium-fixed material. In this report an attempt is made to identify the particulate component by cytochemical means, and a method is described by which the particles can be differentially stained for electron microscopy. It forms an extension of earlier work in which brief reference was made to the problem of cytoplasmic particles in amphibian embryos, and to the staining method (Perry & Waddington, 1966). MATERIALS AND METHODS Embryos of Xenopus laevis, Discoglossus pictus and Triturus alpestris at the gastrula, neurula and tail-bud stages of development were selected for the cytochemical tests, as these early embryonic stages were known to contain an abundance of the cytoplasmic particles. The embryos were fixed in: (i) 1 % veronal-acetate buffered osmium 17 Cell Sci. 2

2 258 M. M. Perry tetroxide with sucrose (Caulfield, 1957) for 2^h; (ii) 2-5% phosphate-buffered glutaraldehyde for 4 h, rinsed overnight in the buffer and post-fixed in osmium tetroxide for 2h (Sabatini et al. 1963); or (iii) 2-5% phosphate-buffered glutaraldehyde only, for 4 h. After dehydration in a graded series of alcohols, pieces of the embryos which had been excised either during or after fixation were embedded in Araldite. Some were dehydrated and embedded in glycol methacrylate (Leduc, Marinozzi & Bernhard, 1963). Thin sections were cut on an LKB Ultratome and mounted on carbon-formvar coated grids. For light microscopy, sections approximately 0-5 /i thick were cut on the same microtome and mounted on albumen-coated slides. Thick sections of various combinations of fixative and embedding media were subjected to the periodic acid/schiff (PAS) test for polysaccharides (Munger, 1961). Thin sections from the same blocks were stained either with a concentrated solution of lead citrate (Reynolds, 1963) or with a saturated, aqueous solution of uranyl acetate followed by lead citrate, for observation in the electron microscope. In order to verify the results of the PAS reaction, enzymic hydrolysis was performed on material embedded in glycol methacrylate for both light and electron microscopy. Thick sections were incubated with salivary amylase for 1 h, or for 30 min with a-amylase (twice crystallized, Worthington Biochemical Corporation) diluted with water, as recommended by Paegle (1964), to obtain an approximate concentration of 0-2%, or with water only as a control. They were then treated with PAS. Thin sections (silver to gold in colour) were incubated in the following solutions: 0-02% a-amylase for 30 min; o-i M phosphate buffer for 2 h; distilled water for 2 h. The incubation temperature in all cases was 38 C. The sections were then stained with lead citrate, or with uranyl acetate and lead citrate. In addition to these treatments, some thin sections were oxidized with periodic acid, rinsed with distilled water, dried and then stained with lead citrate for min (Perry & Waddington, 1966). The concentrations of periodic acid used were, for Araldite, c-8-3'2% for 30 min; for glycol methacrylate, 0-2% for 10 min. The sections were examined with an AEI EM6 electron microscope. RESULTS Electron microscopy Material which had been fixed in osmium tetroxide and embedded in Araldite showed considerable variation both in the number of particles present in the cytoplasm and in their size, apart from the large yolk, lipid and pigment granules. In many cases they were sparsely scattered in the cells, and ranged in diameter from 170 to 300 A. In some fixation batches, particularly of anuran gastrulae, the particles were preserved in fairly large numbers (Fig. 1). At the tail-bud stage, when some elements of endoplasmic reticulum had been formed, the attached ribosomes were 170 A in diameter, and the free particles in the surrounding cytoplasm varied in size. Double-fixed material embedded in Araldite presented a more consistent picture. The 'background' cytoplasm was filled with particles which were of two sizes only, approximately 175 A (± 25 A) and 325 A (± 50 A) in diameter, the larger being pre-

3 Glycogen in amphibian embryos 259 dominant, especially in the earlier stages (Fig. 2). There was little variation in the sizes of the two types of particle between the three species examined. In Discoglossus gastrulae most of the larger particles were closely associated with each other, and occupied all the available space between other organelles. Sometimes they were seen in the intercellular spaces. The smaller particles were often located around the nucleus and appeared in loose clusters in other regions of the cytoplasm. At later stages in both Discoglossus and Xenopus the smaller particles were to be found, for the most part, attached to the membranes of the endoplasmic reticulum, whereas the larger particles remained free in the cytoplasm (Fig. 5). Figure 2 illustrates an intermediate stage with both free and attached 175-A particles. With lead staining alone or with double staining, the larger particles were more electron dense than the smaller, and when examined at high magnification showed an indistinct stippled appearance (Fig. 7), which could be attributed either to a substructure within the particles or to a general graininess in the specimen, perhaps caused by the structure of the supporting film. Light microscopy When nearly contiguous sections of the osmium-fixed, Araldite-embedded materia described in the preceding section were treated by the PAS method it was found that those areas containing large numbers of fine cytoplasmic particles stained intensely, and those containing few particles stained faintly or not at all. Nuclei and yolk granules produced a negative reaction. Figure 1 (inset) shows a PAS-treated section of cells in which the cytoplasmic particles have been well preserved, as judged by the electron microscope image of a nearby thin section (Fig. 1). There is some variation in the intensity of the staining reaction from cell to cell. This could be correlated with the concentration of particles in the corresponding thin section. Double-fixed material gave similar results. Enzyme digestion Amylase digestion of polysaccharides has been attempted to date at two points in the preparative process for electron microscopy. It has been employed either on sectioned, Epon- or Araldite-embedded material, with variable results, or on the fixed tissue blocks, prior to embedding, with more satisfactory results (Coimbra & Leblond, 1966; Lentz, 1966). With the introduction of the water-soluble embedding medium, glycol methacrylate by Leduc et al. (1963) it is now possible to carry out enzyme digestion of cellular components in sectioned material, thus facilitating the control of the process and the analysis of results. Gastrulae were selected for this experiment as they contained considerable quantities of the larger particles. Fixation in glutaraldehyde alone and embedding in glycol methacrylate resulted in an almost complete extraction of cytoplasmic particles, and a corresponding loss in intensity of the PAS reaction. Double-fixation on the other hand gave better preservation of the cytoplasmic components; that is, there were no ' empty spaces' in the cells. However, the picture differed from that obtained with Aralditeembedded material. The bulk of the cytoplasm was occupied by material which occurred either as discrete particles of indeterminate size, or as large clumps of aggre- 17-2

4 260 M. M. Perry gated particles, or in amorphous masses (Fig. 3). Sometimes this material was also seen in the intercellular spaces. Small particles, approximately 175 A in diameter, appeared as discrete bodies situated around the nucleus and in loose clusters in other parts of the cytoplasm. When thin sections were incubated with a-amylase the polymorphic component was removed entirely, leaving holes in the section (Fig. 4), and other structures, including the 175-A particles, remained intact. The component was retained in sections incubated in water and phosphate buffer. Prolonged incubation, of 3-4 h in any medium, resulted in either fading or total extraction of the polymorphic component. In Fig. 4 it is of interest to note that some 175-A particles are arranged in a helical polysomal-type configuration, as has been described in a number of embryonic cells (Behnke, 1963; Waddington & Perry, 1963), and some are in double linear arrays suggestive of rough-surfaced endoplasmic reticulum, although the membranes have not been preserved. It therefore seems likely that the free 175-A particles are ribosomal. Control experiments for light microscopy showed intense PAS-positive regions in sections incubated in water, and some diminution, but not complete abolition, of the staining reaction in sections incubated in amylase. Selective staining for electron microscopy In order to determine more precisely the nature of the particles an attempt was made to stain them selectively. It is well known that glycogen has an affinity for lead (Watson, 1958) but this heavy metal also enhances the contrast of other cytoplasmic structures. Marinozzi (1963), however, has reported that it is possible to stain selectively for glycogen and ribosomes with a lead solution if the bound osmium is first removed from tissue sections by oxidation with hydrogen peroxide or periodic acid. Tail-bud stages of double-fixed, Araldite-embedded material were selected initially for this test, as the ribosomes could be clearly recognized from their location on the endoplasmic reticulum. Periodic acid was employed in preference to hydrogen peroxide for convenience. When sections were oxidized in this manner and subsequently stained with lead citrate, the cellular components showed little contrast, with the exception of the 325-A particles which stained intensely (Fig. 6). The extent to which the other structures stained depended on the concentration of periodic acid used. Closer examination of the densely stained particles showed that they were composed of subunits, approximately 40 A in diameter (Fig. 8). Conversely, the ribosomes on the endoplasmic reticulum were less intensely stained and showed no subunit structure (Fig. 6). This staining method produced comparable results in all three species examined, regardless of whether the tissue had been fixed in osmium tetroxide alone (Fig. 9) or double-fixed. Furthermore, when glycol methacrylate sections were subjected to a similar treatment the aggregated and amorphous component described above showed evidence of a granular structure (Fig. 10), which was not apparent in control sections stained with lead only. In this case over-oxidation of the sections caused complete extraction of the component. The use of uranyl acetate, in addition to lead, to stain the oxidized sections tended to obscure the fine detail in the particles.

5 Glycogen in amphibian embryos 261 DISCUSSION The corroborative evidence obtained from the PAS reaction and the enzyme digestion experiments indicates that large areas of the cytoplasm in embryonic amphibian cells are occupied by glycogen. Although satisfactory digestion was obtained on glycol methacrylate sections, it was found that the extractable material was only infrequently in the particulate form observed in the Araldite sections. However, Revel (1964) has reported that embedding in methacrylate can cause clumping of particulate glycogen. It is concluded therefore that the abundant 325-A particles observed in the doublefixed, Araldite-embedded material are indeed glycogen. In a study of glycogen obtained from fractionated liver Drochmans (1962) has shown that this substance can be degraded progressively, by reducing the ph of the medium, from alpha particles ( m/i) to beta particles (20-30 m/i), and ultimately to gamma elements (3 m/i in diameter, 20 m/i in length). Revel (1964) and Berthold (1966) have demonstrated a subunit structure or stippling effect in glycogen particles in liver and frog spinal ganglia. Murdoh, Leloir & Krisman (1965) have shown that during in vitro synthesis of glycogen, particles m/i in diameter are formed, and that these particles are made up of m/i subparticles. It is probable that the 325-A particles observed in amphibian embryos correspond to the beta glycogen of Drochmans. However, in the majority of preparations gamma-type elements were not detectable. Only when the sections were treated with periodic acid/lead did a substructure of 40-A units become clearly evident. Whether these subunits are identical to or reflect the same molecular substratum as those obtained by other workers is uncertain. Ribosomal elements could be identified as such only from their location on the endoplasmic reticulum at the later developmental stages. At earlier stages there was little or no endoplasmic reticulum to enable one to ascertain the identity of the smaller, 175-A particles. Attempts to digest them with ribonuclease gave no further clue as to their identity, but this is not necessarily significant for Leduc et al. (1963) have pointed out that prolonged fixation, especially in osmium tetroxide, renders substances impervious to enzyme digestion and it is probable that in the present case the material was too well fixed for effective digestion of ribonucleic acids to take place. Since the size of the free 175-A particles in the gastrula and neurula stages corresponded to the ribosomes on the endoplasmic reticulum and to the helical polyribosomes, it is assumed that they too are ribosomes. Amongst the methods available for the selective staining of glycogen may be mentioned the periodic acid/silver methanamine reaction (Movat, 1961) and the hydrogen peroxide/lead stain (Marinozzi, 1963). These methods suffer from the disadvantage that some other cell constituents also have their contrast enhanced by the stain. Neither is reported to show up a substructure in the glycogen particles. According to Revel (1964), the most satisfactory method of identifying glycogen, and at the same time of distinguishing it from ribosomes, is to fix the material in potassium permanganate, thereby eliminating the ribosomes, and then to stain the sections with lead. Minio, Lombardi & Gautier (1966) report that the presence of a clear zone around the

6 262 M. M. Perry glycogen allows a distinction to be made between the two components. In the present investigation it was found that by treating the sections with periodic acid/lead citrate it was not only possible to increase, selectively, the electron density of the glycogen particles, but also to demonstrate a subunit structure in these particles, in contrast to the ribosomes. There is therefore reason to believe, on purely empirical grounds, that the method is specific for glycogen in amphibian embryonic tissue. Recent experiments have shown that it is also applicable to glycogen deposits in the eye of Drosophila (unpublished observations). It offers several advantages over existing methods in that it can be employed routinely on material preserved in a number of ways, and that other cell constituents can be seen simultaneously, despite their comparative lack of contrast. It is particularly valuable in distinguishing particulate glycogen from ribosomes at early development stages, when the ribosomal population also seems to be in the form of free particles. REFERENCES BEHNKE, O. (1963). Helical arrangement of ribosomes in the cytoplasm of differentiating cells of the small intestine of rat foetuses. Expl Cell Res. 30, BKRTHOLD, C. H. (1966). Ultrastructural appearance of glycogen in the /J-neurons of the lumbar spinal ganglia of the frog. J. Ultrastruct. Res. 14, CAULFLELD, J. B. (1957). Effects of varying the vehicle for OsO«in tissue fixation. J. biophys. biochem. Cytol. 3, COIMBRA, A. & LEBLONB, C. P. (1966). Sites of glycogen synthesis in rat liver cells as shown by electron microscope radioautography after administration of glucose-h 1. J. Cell Biol. 30, i5i-i75- DROCHMANS, P. (1962). Morphologie du glycogene. Etude au microscope electronique de colorations negatives du glycogene. J. Ultrastruct. Res. 6, EAKIN, R. M. (1964). Actinomycin D inhibition of cell differentiation in the amphibian sucker. Z. ZeUforsch. mikrosk. Anat. 63, GREGG, J. R. (1948). Carbohydrate metabolism of normal and of hybrid amphibian embryos. J. exp. Zool. 109, HEATLEY, N. G. & LINDAHL, P. E. (1937). Distribution and nature of glycogen in the amphibian embryo. Proc. R. Soc. B 12a, JAEGER, L. (1945). Glycogen utilisation by the amphibian gastrula in relation to invagination and induction, y. Cell. comp. Physiol. 25, LEDUC, E., MARINOZZI, V. & BERNHARD, W. (1963). The use of water-soluble glycol methacrylate in ultrastructural cytochemistry, yi R. microsc. Soc. 81, LENTZ, T. L. (1966). Intramitochondrial glycogen granules in digestive cells of Hydra, y. Cell Biol. 29, MARINOZZI, V. (1963). The role of fixation in electron staining, yi R. microsc. Soc. 81, MINIO, F., LOMBARDI, L. & GAUTIEH, A. (1966). Mise en evidence et ultrastructure du glycogene hepatique. Influence des techniques de preparation, y. Ultrastruct. Res. 16, MOVAT, H. Z. (1961). Silver impregnation methods for electron microscopy. Am. y. clin. Path. 35, MUNGER, B. L. (1961). Staining methods applicable to sections of osmium fixed tissue for light microscopy, y. biophys. biochem. Cytol. 11, MURDOH, J., LELOIR, J. F. & KRISMAN, C. R. (1965). In vitro synthesis of particulate glycogen. Proc. natn. Acad. Set. U.S.A. 53, PAEGLE, R. D. (1964). Histochemical study of contaminants in commercially available diastases and amylases. y. Histochem. Cytochem. 12, PERRY, M. M. & WADDINGTON, C. H. (1966). The ultrastructure of the cement gland in Xenopus laevis. y. Cell Sci. 1, REVEL, J. P. (1964). Electron microscopy of glycogen. y. Histochem. Cytochem. 12,

7 Glycogen in amphibian embryos 263 REYNOLDS, E. S. (1963). The use of lead citrate at high ph as an electron-opaque stain in electron microscopy. J. Cell Biol. 17, SABATINI, D. D., BENSCH, K. & BARRNETT, R. J. (1963). The preservation of cellular ultrastructure and enzymatic activity by aldehyde fixation. J. Cell Biol. 17, WADDINGTON, C. H. & PERRY, M. M. (1963). Helical arrangement of ribosomes in differentiating muscle cells. Expl Cell Res. 30, WATSON, M. L. (1958). Staining of tissue sections for electron microscopy with heavy metals. J. biophys. biochem. Cytol. 4, {Received 26 November 1966)

8 264 M. M. Perry ABBREVIATIONS g I n r' glycogen lipid droplets nucleus small particles, probably ribosomes P r y pigment granules ribosomes yolk granules Fig. 1. Discoglossus gastrula, dorsal region. Inset, photomicrograph of a section stained with PAS; x The cytoplasm of the centrally placed cell shows a moderately intense staining r< action, the nucleus, yolk granules and lipid granules have not stained. The electron micrograph, of a nearby thin section of the area outlined, shows yolk granules, lipid droplets, pigment granules, mitochondria and a fine particulate material in the cytoplasm. Osmium tetroxide, Araldite, lead stain, x Fig. 2. Discoglossus neurula, cement gland. Fine particles of two distinct sizes occupy most of the cytoplasm, the larger (g) are glycogen, the smaller, attached to the endoplasmic reticulum, are ribosomes (r). Similar small particles grouped near the nucleus are probably also ribosomes (r'). Double-fixation, Araldite, lead stain, x

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10 Fig. 3. Discoglossus gastrula, dorsal region. Much of the cytoplasm is occupied by a component consisting of aggregated particles (arrows). Small, discrete particles (r r ) appear around the nucleus, and in groups elsewhere. Double-fixation, glycol-methacrylate, double-staining, x 32coo. Fig. 4. As Fig. 3, to show the effect of amylase digestion. The aggregated component has been extracted. The remaining, discrete particles (r') are mostly free in the cytoplasm, and some are in helical or linear configurations characteristic of ribosomes (r). x

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12 Fig. 5. Xenopus tail bud, cement gland. The larger, dense particles, which are free in the cytoplasm are glycogen, the small particles attached to the endoplasmic reticulum are ribosomes. Double-fixation, Araldite, double-staining, x Fig. 6. As Fig. 5. Section treated with periodic acid and stained with lead. The larger glycogen particles have stained intensely and are composed of subunits, the smaller ribosomes are less intensely stained, and have no substructure. Doublefixation, Araldite. x

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14 Fig. 7. Triturus gastrula, blastopore cell. The glycogen particles have a faintly stippled appearance. Double-fixation, Araldite, double-staining, x Fig. 8. As Fig. 7, to show the clearly defined structure of subparticles in the glycogen resulting from periodic acid/lead treatment, x Fig. 9. Xenopus neurula. Periodic acid/lead. Most of the cytoplasmic particles show the characteristic subunit structure of glycogen. Osmium tetroxide, Araldite. x Fig. 10. Discoglossus gastrula. Glycol-methacrylate embedded (compare with Fig. 3), to show the appearance of the aggregated glycogen particles when treated with periodic acid/lead, x

15 Journal of Cell Science, Vol. 2, No. 2 '.%-' 4 «> a % X r ^i V T, Q-V yjf TI M. M. PERRY

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