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Fasciola hepatica: An electron microscope autoradiographic study of protein synthesis and secretion by gut cells in tissue slices
EXPERIMENTAL
38, 167-180
PARASITOLOGY
( 1975)
fasciola hepatica: An Electron Microscope Study of Protein Synthesis and Secretion in Tissue Slices
Autoradiographic by Gut Cells
R. E. B. HANNA ’ Zoology Department, (Accepted
Queen’s University,
for publication
January
Belfast 2, 1975)
HANNA, R. E. B. 1975. Fmciola hepatica: An electron microscope autoradiographic study of protein synthesis and secretion by gut cells in tissue slices. Experimental Parasitology 38, 167-180. A loop technique for electron microscope autoradiographic studies on slices of Fusciola hepatica is described, and used to study the synthesis of protein-containing bodies by gut cells of the fluke. Slices were pulse labeled with tritiated tyrosine, methionine, leucine, and phenylalanine, and, after appropriate chase periods in unlabeled medium, were fixed with formaldehyde and prepared for electron microscopy by a procedure involving ethylene glycol dehydration. Labeled amino acids were found to be incorporated into protein, or glyoprotein, in the gut cells of the slices by a GER-Golgi complex mediated mechanism similar to that which occurs in vertebrate tissues. The precursors appeared to enter the cells across their basal and lateral plasma membranes and mature secretory bodies were discharged at the cell apex. INDEX DESCRIPTORS: Fascioh hgatica slices; Electron microscope autoradiography; [‘HI-tyrosine; [3H]-methionine; [“HI-leucine; [“HI-phenylalanine; Pulse-chase technique; Ethylene glycol dehydration; Loop technique; Protein synthesis; Gut cells; GER-Golgi system; Eccrine secretion; Metabolism.
A large number of morphological studies have shown that certain cellular activities occur in Fasciola hqatica, and other trematodes and cestodes, which might be further investigated using light and electron microscope tracer techniques. In fact, many such studies have already been undertaken (Bjorkman and Thorsell 1964; Burton 1962; Lumsden 1966; Nollen 1968; Oakes and Lumsden 1971; Pappas 1971; Robinson 1971; Shannon and Bogitsh 1971; Thorsell, Bjorkman, and Appelgren 1966; Thorsell, Appelgren, and Kippar 1968). 1 Present address: E.A.V.R.O., Box 32, Kikuyu, Kenya.
Muguga,
P.O.
167 Copyright All rights
@ 1975 by Academic Press, Inc. of reproduction in any form reserved.
At present, there is only one published account of the use of electron micro’scope autoradiography using helminth material, (Shannon and Bogitsh 1971). In this study it was shown that [3H]-galactose was incorporated into rod-shaped bodies in the Golgi region of the tegumental cells of Megalodiscus temperatus. These bodies subsequently passed to the surface syncytium via connecting tubules, and their contents contributed to the glycocalyx which coats the outer as’pect of the apical plasma membrane. Interestingly, incorporation occurred when slices of the worm were incubated in radioactive precursors, but not when whole worms were used. This suggests that labeled galactose was ac-
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tively cxcludcd from the internal cells and tissues of the intact worm. The slice technique described in previous reports (Hanna and Threadgold 1975; Threadgold and Hanna 1975), was designed to allow test s~rbstances, such as metabolic inhibitors and radioa’ctive precursors, access to cells and organ systems of F. hepatica not normally in contact with the environment. Thus it should be ideally suited to autoradiographic studies such as that detailed above. The main emphasis in the present work was on the development of a technique which could be used routinely for electron microscope autoradiographic studies of F. hepatica. The results which will be described relate to the incorporation of amino acids by the epithelial cells of the gut of slices of the fluke and to the subsequent migration and discharge of the labeled secretory bodies. The morphology of the epithelial cells of the gut of F. hepatica was described by Robinson (1971), who proposed that they synthesise protein by a GER-Golgi mediated mechanism similar to that which occurs in vertebrate cells (Caro and Palade 1964; Essner and Novikoff 1962; Kurosumi 1961; Siekevitz 1959). An experimental investigation of this proposition is presented here. MATERIALS
AND METHODS
Incubation, labeling, and tissue preparation. Labeling was carried out by a pulsechase technique in which slices of mature flukes from rats, collected and prepared as described by Hanna and Threadgold (1975), were incubated for a brief pulse period in medium containing a tritiated amino acid and were then given various chase periods in unlabeled medium prior to fixation. Ml99 (pH 7..5 at 37 C, 290 mOsM, with benzylpenicillin ( sodium), 2000 IU/ml, and streptomycin sulphate, 2 mg/ml), was used for both pulse and chase media, but it was considered advisable to eliminate the unlabeled form of
each particular precursor frour the pulse medium, before adding the labeled precursor. This was to ensure that, of the precursor molecules used, as many as possible were labeled, since the greater the amount of radioactivity incorporated in the tissm the more intense is the autoradiographic reaction. Therefore, a special consignment of dried Ml99 was obtained from the manufacturer, Wellcome Research Laboratories, Reckenham, which was deficient in those substances whose incorporation was to bc investigated, namely phenylalanine, methionine, leucine, and tyrosine. When the labeled pulse medium was prepared for an experiment, all these constituents were replaced in the recommended amounts, and in the unlabeled form, except for the one amino acid which was to be studied and was in the labeled form. Tritiated amino acids were obtained from the Radiochemical Centre, Amersham, and their concentration in the experiments was kept as high as possible (100 &/ml) by using small volumes of pulse medium (1.0 ml). Prior to each experiment the fluke slices were rinsed in unlabeled Ml99 (at 37 C) which lacked the amino acid under study. Several slices were fixed in 10% formaldehyde in Millonig’s buffer pH 7.4, to act as unlabeled controls, and the remainder were placed for 5 min in the pulse chamber (Fig. l), with 1.0 ml of labeled medium. The temperature was maintained at 37 C by a heating jacket (J, Fig. 1) through which water was circulated, and a fine stream of gas (95% Na, 5% COz), was passed through the medium, providing gentle agitation and an anaerobic environment. After pulsing, the slices were rinsed in warm chase medium containing the test molecule in unlabeled form, and several slices were fixed immediately as for the controls. The remaining slices were incubated in chase medium, at 37 C under
AUTORADIOGRAPHY
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PROTEIN
SYNTHESIS
IN
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FIG. 1. Incubation chamber and connections. G, rubber hose to 95% N2/5% CO, gas supply; B, water-filled chamber through which gas is bubbled; H, hypodermic needles, V, vent for incubation chamber; I, incubation chamber surrounded by a jacket, J, through which warm water circulates in the direction of the white arrow.
anaerobic conditions with rocking, and several slices fixed after 5, 10, 20, 30, 45, and 60 min. All the slices were fixed for 24 hr at 4 C in 10% formaldehyde in Millonig’s buffer, pH 7.4. This fixative was used instead of glutaraldehyde because the latter binds unincorporated amino acids to structural proteins by cross-linking, and, therefore, gives fixation artifacts in autoradiograms (Hodgson and Marshall 1967; Peters and Ashley 1967, 1969), Formaldehyde is a satisfactory fixative for electron microscopy (Baker and McCrea 1966) and does not give this artifact (Peters and Ashley 1967, 1969). After fixation, the slices were washed in Millonig’s buffer, and post fixed in 1% osmium tetroxide in Millonig’s buffer, pH 7.4. The slices were then dehydrated by an ethylene glycol series, 20, 50, 75, and 90% for 30 min each, then 100% for 24 hr with frequent changes, passed through pro-
pylene oxide (4 hr) and embedded in Araldite. This procedure was adopted after several unsuccessful experiments had been carried out using alcohol as the dehydrant. It is possible that alcohol extracts considerable amounts of protein and glycoprotein from tissue fixed in formaldehyde and it is known that “inert” dehydrants, such as ethylene glycol, retain many low molecular weight substances in substantially unaltered quantities (Pease 1965; Sjostrand 1971). Preparation for electron microscope autoradiography. The techniques and problems of electron microscope autoradiography have been critically reviewed and discussed by many authors including Caro (1962, 1966), Caro and van Tubergen (1962), Granboulan ( 1965), Jacob ( 1971), Pelt ( 1963), Salema ( 1970), and Salpeter and Bachmann ( 1965). The method used in this investigation was based on the “loop”
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HANNA
AUTORADIOGRAPHY
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technique of Caro and van Tubergen ( 1962), which is one of the simplest techniques available and which has been used successfully in ‘a number of important studies (Caro and Palade 1964; Jamieson and Palade 1967b, 1968a, b; Shannon and Bogitsh 1971). UItrathin sections were cut from the slices embedded in Araldite using an LKB III ultramicrotome and mounted on Formvar-coated grids of nickel. The coating procedure was carried out under darkroom conditions using a Kodak minimum red safelight mounted about 3 ft from the working area. The emulsion solution was prepared in a Coplin jar by dissolving 10 g of Ilford L4 nuclear research emulsion (gel form) in 20 ml of distilled water at 45 C. The mixture was maintained at 45 C in a waterbath for 15 min and was frequently stirred with a paraffin coated glass rod, The emulsion solution was then immersed in an ice bath for 2 min, stirred, and kept at room temperature for a further 30 min, with occassional stirring, before use. For coating each grid was balanced (section side upward), on a wooden peg (3 mm diam), a number of which were mounted vertically on a baseboard. A film of gelled emulsion was formed across a Nichrome wire loop by dipping the latter into emulsion, withdrawing it gently, and holding it with the film vertical for 5 sec. The film was then applied to the grids by bringing the loop down briskly over the peg, at an angle of 45”, completely to the baseboard. The film generally burst within seconds leaving a thin layer of emulsion
SYNTHESIS
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adhering to the surface of the grid and sections. After coating, each grid was stuck by its edge ‘and with the sections uppermost to a strip of double-sided sellotape mounted on a clean glass slide. Several grids were stuck to each slide and when two slides were completed, they were labeled appropriately on their free surfaces with a black wax pencil and placed in plastic two slide containers with the grids facing outward. The space between the slides was packed with silica-gel crystals and the container sealed and labeled. Each container had previously been made light-tight with black insulating tape ‘and enamel paint. The containers associated with each experiment were packed together in a light- and air-tight box, with silica-gel, and were stored at 4 C for 5 mo. After the exposure period, the grids were gently removed from the slides and developed at 20 C in Kodak D-19 developer (2 min). Following a rinse in 1% acetic acid, the preparation was fixed in Ilford “Hypam” fixer ( 1 in 10 dilution for 5 min), and washed for 30 min in distilled water which was changed frequently. Processing was carried out in the depressions of white porcelain tiles and in embryo dishes and it was found that cocktail sticks and slightly magnetic forceps were convenient for manipulating the grids. Prior to electron microscope examination, the autoradiograms were treated with an aqueous solution of 0.2% sodium hydroxide for 90 set, in order to remove the gelatin of the photographic emulsion. The sections were then stained for 5 min with
FIG. 2. i3Hltyrosine incubation; 5 min pulse; 5 min chase. Silver grains (white arrows) are present at the base of the gut epithelial cells (B) and the basal granular endoplasmic reticulum, (ER). Activity is also associated with muscle blocks (MU) in the thick layer of interstitial connective tissue (I). X25,000. FIG. 3. [“Hltyrosine; 5 min pulse, 5 min chase. Silver grains are present over the granular endoplasmic reticulum (ER), adjacent to the lateral plasma membranes of the cells (L). Note that the membranes of the ER cistemae appear as negative images (“white” lines), and the cisternal contents as a “dark” line. M, mitochondrion showing a negative image of its outer, inner, and qristal membranes. X40,000,
FIG. 4. [3H]tyrosine; 5 min pulse, 20 min chase. Grains are present in the vesicular component (GV) of the Golgi complex and in adjacent ER, condensing vacuoles (CV), and secretory bodies ( S) . M, mitochondrion. ~40,000. FIG. 5. [3H]tyrosine; 5 min pulse, 20 min chase. Silver grains are associated mainly with secretory bodies (S), but some also lie over the endoplasmic reticulum (ER). N, nucleus. x34000. 172
FIG. 6. [3H]tyrosine; 5 min pulse; 30 mm chase. The cell apex is shown and silver are associated with secretory bodies (S), Golgi vesicles (GV), and the apical lamellae which project from the cell surface into the lumen. ~25,000. FIG. 7. [‘Hltyrosine; 5 min pulse, 30 min chase. Grains overlie a condensing vacuole in the apical part of the cell. LA, lamellae. X65,000. FIG. 8. [$H]methionine; 5 min pulse, 30 mm chase. A silver grain lies over a secretory ( S ) in the process of being extruded into the lumen of the gut. LA, lamellae. X62,500. 173
grains (LA) (CV) body
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E.
B.
HANNA
AUTORADIOGRAPHY
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alcoholic uranyl acetate and for 4 min in lead citrate. The unincubated control material was coated with photographic emulsion and subsequently treated for autoradiography along with the incubated material. The sections of material from the controls showed no labeling over the gut cells. RESULTS
All four amino acid tracers ( [3H]tyrosine, [ 3H]methionine, [ 3H] leucine, and [ 3H]phenylalanine), were found to be incorporated by the epithelial cells of the gut and the distribution of the label within the cells varied with the length of the chase period. The structures of the cells over which silver grains occurred were the GER, the Golgi complexes, the secretory bodies, and the apical lamellae, and, regardless of chase period, labeled cells showed activity over most of these sites. However, the frequency with which grains occurred over the various sites was dependent on the length of the chase period. With material which had been given short chase periods (O-10 min), most of the silver grains were found over the GER, particularly that near the base of the cells and adjoining the lateral plasma membranes (Figs. 2 and 3). After a longer chase period (20 min), much of the activity was located at or near the Golgi complexes. Grains were observed over the GER adjoining the Golgi, over the small transition vesicles between the GER and the Golgi, and over the large secretory bodies arising from the maturing face of the Golgi complex (Figs. 4 and 5). In material which had been given 30 or 45 min chase periods, labeling was predominantly in the apical regions of the cells and was associated with
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the secretory bodies (Figs. 6 and 7)) with discharging secretory material at the cell surface (Fig. S), or between the lamellae (Fig. 9), and with the lamellae themselves (Fig. 10). More activity was present over the lamellae after 45 min chase than after 30 min, and one autoradiogram of the former material showed labeling associated with flocculant material in the gut lumen (Fig. 11). Little activity was detected in material which had received a 60-min chase period. In order to check that the localization of activity in labeled cells was influenced by the length of the chase period, the distribution of silver grains in a series of autoradiograms of [3H]methionine labeled slices was analysed. This was done by noting in each autoradiogram the ultrastructural localization of the silver grains. The labeled sites were classified into the following groups; GER, GER associated with the Golgi complex, Golgi complex, secretory bodies at the cell apex, and lamellae. For each chase period a total of 200-300 grains in two or three autoradiograms was recorded and the number of grains lying over each of the five ultrastructural categories expressed as a percentage of the total count (Fig. 12, Table 1). The results are illustrated in Fig. 12, which shows that immediately after labeling most activity is over the GER and that during the succeeding chase periods a “wave” of activity passed through the cells. The labeled material moved from the GER to the Golgi and eventually reached the apex of the cells in the secretory bodies. From the apex it was discharged and became associated with the lamellae. The movement was not fully synchronised, however, and a certain amount -
FIG. 9. [‘Hlphenylalanine; 5 min pulse, 45 min chase. A grain lies over a number of lamellae and near a secretory body (S), Th e 1amellae appear as dense cores of cytoplasm (C) flanked by negative images of membranes and an outer fuzzy coat. X120,000. FIG. 10. [“Hltyrosine; 5 min pulse, 30 min chase. Grains are associated with lamellae (LA) and flocculant material in the lumen (white arrow). X75,000. FIG. 11. [3H]tyrosine; 5 min pulse, 45 min chase. A silver grain (white arrow) lies over flocculant material in the gut lumen. X40,000.
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c 0
E.
B.
HANNA
12 10
20
JO
45 min
FIG. 12. Incorporation of [‘Hlmethionine into gut epithelial cells. The graphs show the % of incorporation with time into different organelles and structures. ER, granular endoplasmic reticulum; ER/GC, boundary between the endoplasmic reticulum and the forming face of the Golgi complex; GC, maturing sacs of the Golgi complex; S, secretory bodies; LA, lamellae.
of activity was left behind at each ultrastructural location. TABLE Distribution [“HI
of Siluer Methionine Cells,
After
yO Total
Location
GER GEIt/Golgi Golgi Secretory bodies Lamellae --.--.-
I
Grains in Autoradiograms Pulse-La&led Epithclial Various Chase Periods
___ ~--
grains periods
of
after chase of ____
0 min
10 min
20 min
30 min
45 min
60 13 9
43 26 14
22 18 24
20 11 28
12 4 27
10 8
13 4
19 17
27 14
31 26
DISCUSSION
Protein synthesis by the epithelial cells. The results of the experiments with tritiated amino acids show clearly that the GER and Golgi complexes of the epithelial cells of the gut of Fasciola hepotica are involved in the synthesis of protrin-containing material, which is subsequently discharged into the gut lumen. The mechanism involved is similar to that which has been demonstrated by autoradiographic methods for vertebrate cells (Caro and Palade 1964; Jamieson and Palade 1967a; 1968a, b, c), and it agrees closely with
AUTORADIOGRAPHY
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the mechanism proposed by Robinson ( 1971)) on morphologic21 evidence. The common occurrence of activity in the GER near the base and lateral margins of the cells in material from short chase periods, suggests that the labeled amino acids entered across the basal and lateral membranes, rather than across the apical membrane. The interstitial material may have a role in bringing precursors within the range of the transport membranes since it occurs as a thick layer immediately beneath the basal lamina of the cells. On the other hand the inpushings of the parenchymal cells into the base of the gut epithelial cells may be a more important region of transfer and the junctional complexes between parenchymal and gut cells may be the sites of precursor transport and are known to contain acid and alkaline phosphatases (Threadgold 1968; Gallagher and Threadgold 1967; Robinson 1971) . This problem might be resolved by electron microscope autoradiography with frozen or freeze-dried sections. Since, after the shortest chase periods, most of the activity was confined to the GER, this region must be the first in which amino acids are incorporated into nondiffusable molecules. This is identical to the situation in vertebrate tissues (Caro and Palade 1964; Redman, Siekevitz, and Palade 1966), and suggests that the mechanism for protein synthesis which operates in F. hepatica is very similar to that in vertebrates, i.e., manufacture of polypeptide and protein chains at the ribosomal sites, with subsequent passage of the completed molecules into the GER cisternae for transport within the cell. The concentration of activity in the GER adjacent to Golgi complexes, after a lo-min chase, suggests that the labeled protein was transported to this region in the GER cisternae. Subsequently the activity appeared in the Golgi complexes themselves, and was associated with the small pe-
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ripheral vesicles which bud off from the GER (Robinson 1971) and with the large secretory vesicles. This suggests that the small vesicles (transition vesicles) are vehicles for the transfer of secretory proteins from the GER to the condensing vacuoles (secretory vesicles ) of the Golgi complexes. Thus these vesicular components are probably analogous to the vesicular structures present around and within the Golgi complexes of vertebrates (Jamieson and Palade 1967a, b). The labeled condensing vacuoles were found to concentrate toward the apices of the cells after long chase periods and formed into clusters. Discharge of these labeled secretory bodies was effected by eccrine secretion (Kurosumi 1961). In this process the limiting membrane of the secretory body fuses with the apical membrane of the cell, hence becoming part of the membrane of the lamellae, and the contents are released onto the surface. The frequent occurrence of activity on the lamellae of the epithelial cells after long chase periods suggests that some of the labeled amino acids were incorporated into, or intimately bound to the membranes of the secretory bodies, rather than with their contents. It is possible that the inner aspect of these membranes is lined with glycoproteins, which become part of the glycocalyx of the lamellae when the contents of the secretory bodies is discharged. Most of the activity associated with the discharged contents of the secretory bodies was probably lost during processing for electron microscopy, since this material, which is perhaps a proteolytic enzyme, would lie free in the gut lumen. The results show that the movement of radioactivity through the epithelial cells was not entirely synchronous and labeled material was retained at each stage of the synthetic process. This may have been partly due to the incorporation of label into structural protein or cytoplasmic enzymes. A similar phenomenon was ob-
17x
R. E. B. HANNA
served in the goblet cells of the mouse colon by Rhor and Richter (1967), and these authors used the term “sessile protein” to describe the labeled material remaining in cells. The present work has shown that the GER and Golgi complexes in the epithelial cells of the gut of F. hepatica carry out protein or glycoprotein synthesis by a mechanism broadly similar to that which occurs in vertebrate cells. This provides a basis for further detailed studies on protein synthesis in flukes. In particular, the mctabolic requirements of the various stages in the synthetic process could be investigated and the “energy-locks” identified. Studies of this nature have been carried out on vertebrates by Jamieson and Paladc (1967a, b, 1968a, b, c), and a comparison with an invertebrate system would be of great interest. Comment on the clehydmlion technique. Ethylene glycol appeared to preserve many of the molecules which were lost when alcohol was used as the dehydrant, but the ultrastructural appearance of the tissues dehydrated with ethylene glycol was rather unusual. The membranes appeared as “negative images” and dense “lines” were often observed in the centre of GER cisternae. Such “lines” probably represented peptides and proteins which were lost from the cisternal lumen during alcohol dehydration. Large gaps and spaces were common in and between cells, and much of the conventional ultrastructural detail of nuclei and muscle tissue was missing. Sjostrand ( 1971)) found that ethylene glycol dehydration gave particularly good preservation of membranes, but his staining technique (phosphotungstic acid) was different from that used here. Hence it might be possible to improve the appearance of the tissues dehydrated through ethylene glycol by altering the staining procedure. The appearance of gaps and spaces in the tissues may have been due to severe
shrinkage of cells following the transfer to propylene oxide, and this could perhaps be remedied by increasing the concentration of propylene oxide in a gradual manner. With such technical modifications as these to improve the ultrastructural appearance of the tissues, the ethylene glycol dehydration technique should prove useful in further autoradiographic studies on F. lzepaticn and perhaps other helminths. REFERENCES BAKER, J. R., AND MCCREA, J. XI. 1966. The fine structure resulting from fixation by formaldehyde. The effects of concentration duration, and temperature. Journal of the Ro!yal Microscopical Society 85, 391-399. BJOHKMAN, N., AND THORWLL, W. 1964. On the fine structure of the cuticle of the liver fluke, Fasciola hepatica L. Experimental Cell Re.search 33, 319-329. BURTON, P. R. 1962. In zjitro uptake of radioglucose by a frog lung-fluke, and correlation with the histochemical identification of glycogen. Journal of Parasitology 48, 874882. CARO, L. G. 1962. High resolution autoradiography 11. The problem of resolution. Journal of Cell Biology 15, 189-199. CAIIO, L. G. 1966. Progress in high resolution autoradiography. Progress in Biophysics and Molecular Biology 16, 171-190. CARO, L. G., AND PALADE, G. E. 1964. Protein synthesis, storage, and discharge in the pancreatic exocrine cell. An autoradiographic study. Journal of Cell Biology 20, 473-496. CARP, L. G., AND VAN TUBERGEN, R. P. 1962. High Resolution autoradiography. I. Methods. Journal of Cell Biology 15, 173-188. ESWER, E. II., AND NOVIKOFF, A. B. 1962. Cytological studies on two functional hepatomes. Interrelationships of endoplasmic reticulum, Golgi apparatus, and lysosomes. Journal of Cell Biology 15, 189-312. GALLAGHER, S. S. E., AND THREADGOLD, L. T. 1967. Electron microscope studies of Fa.sciola hepatica. II. The interrelationship of the parenchyma with other organ systems. Parasitology 57, 627-632. GIIANBOULAN, P. 1965. Comparison of emulsion and techniques in electron microscope radioautography. Sympos!um of the International Society for Cell Biology 4, 43-63. IIANNA, K. E. B., AND THREAXOLII, L. T. 1975. Development of an in vitro technique for
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after short in citro incubation with some amino acids. ZeitCdzrift fur Parasitenkunde 28, 108-115. THREADGOLD, L. T. 1968. Electron microscope studies of Fasciola hepatica. VI. The ultrastructural localization of phosphatases. Erperimental Parasitology 23, 264-276.
THREADGOLD,
L. T.,
Development cytological hepatica: teria.
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PARASITOLOGY
( 1975)
fasciola hepatica: An Electron Microscope Study of Protein Synthesis and Secretion in Tissue Slices
Autoradiographic by Gut Cells
R. E. B. HANNA ’ Zoology Department, (Accepted
Queen’s University,
for publication
January
Belfast 2, 1975)
HANNA, R. E. B. 1975. Fmciola hepatica: An electron microscope autoradiographic study of protein synthesis and secretion by gut cells in tissue slices. Experimental Parasitology 38, 167-180. A loop technique for electron microscope autoradiographic studies on slices of Fusciola hepatica is described, and used to study the synthesis of protein-containing bodies by gut cells of the fluke. Slices were pulse labeled with tritiated tyrosine, methionine, leucine, and phenylalanine, and, after appropriate chase periods in unlabeled medium, were fixed with formaldehyde and prepared for electron microscopy by a procedure involving ethylene glycol dehydration. Labeled amino acids were found to be incorporated into protein, or glyoprotein, in the gut cells of the slices by a GER-Golgi complex mediated mechanism similar to that which occurs in vertebrate tissues. The precursors appeared to enter the cells across their basal and lateral plasma membranes and mature secretory bodies were discharged at the cell apex. INDEX DESCRIPTORS: Fascioh hgatica slices; Electron microscope autoradiography; [‘HI-tyrosine; [3H]-methionine; [“HI-leucine; [“HI-phenylalanine; Pulse-chase technique; Ethylene glycol dehydration; Loop technique; Protein synthesis; Gut cells; GER-Golgi system; Eccrine secretion; Metabolism.
A large number of morphological studies have shown that certain cellular activities occur in Fasciola hqatica, and other trematodes and cestodes, which might be further investigated using light and electron microscope tracer techniques. In fact, many such studies have already been undertaken (Bjorkman and Thorsell 1964; Burton 1962; Lumsden 1966; Nollen 1968; Oakes and Lumsden 1971; Pappas 1971; Robinson 1971; Shannon and Bogitsh 1971; Thorsell, Bjorkman, and Appelgren 1966; Thorsell, Appelgren, and Kippar 1968). 1 Present address: E.A.V.R.O., Box 32, Kikuyu, Kenya.
Muguga,
P.O.
167 Copyright All rights
@ 1975 by Academic Press, Inc. of reproduction in any form reserved.
At present, there is only one published account of the use of electron micro’scope autoradiography using helminth material, (Shannon and Bogitsh 1971). In this study it was shown that [3H]-galactose was incorporated into rod-shaped bodies in the Golgi region of the tegumental cells of Megalodiscus temperatus. These bodies subsequently passed to the surface syncytium via connecting tubules, and their contents contributed to the glycocalyx which coats the outer as’pect of the apical plasma membrane. Interestingly, incorporation occurred when slices of the worm were incubated in radioactive precursors, but not when whole worms were used. This suggests that labeled galactose was ac-
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tively cxcludcd from the internal cells and tissues of the intact worm. The slice technique described in previous reports (Hanna and Threadgold 1975; Threadgold and Hanna 1975), was designed to allow test s~rbstances, such as metabolic inhibitors and radioa’ctive precursors, access to cells and organ systems of F. hepatica not normally in contact with the environment. Thus it should be ideally suited to autoradiographic studies such as that detailed above. The main emphasis in the present work was on the development of a technique which could be used routinely for electron microscope autoradiographic studies of F. hepatica. The results which will be described relate to the incorporation of amino acids by the epithelial cells of the gut of slices of the fluke and to the subsequent migration and discharge of the labeled secretory bodies. The morphology of the epithelial cells of the gut of F. hepatica was described by Robinson (1971), who proposed that they synthesise protein by a GER-Golgi mediated mechanism similar to that which occurs in vertebrate cells (Caro and Palade 1964; Essner and Novikoff 1962; Kurosumi 1961; Siekevitz 1959). An experimental investigation of this proposition is presented here. MATERIALS
AND METHODS
Incubation, labeling, and tissue preparation. Labeling was carried out by a pulsechase technique in which slices of mature flukes from rats, collected and prepared as described by Hanna and Threadgold (1975), were incubated for a brief pulse period in medium containing a tritiated amino acid and were then given various chase periods in unlabeled medium prior to fixation. Ml99 (pH 7..5 at 37 C, 290 mOsM, with benzylpenicillin ( sodium), 2000 IU/ml, and streptomycin sulphate, 2 mg/ml), was used for both pulse and chase media, but it was considered advisable to eliminate the unlabeled form of
each particular precursor frour the pulse medium, before adding the labeled precursor. This was to ensure that, of the precursor molecules used, as many as possible were labeled, since the greater the amount of radioactivity incorporated in the tissm the more intense is the autoradiographic reaction. Therefore, a special consignment of dried Ml99 was obtained from the manufacturer, Wellcome Research Laboratories, Reckenham, which was deficient in those substances whose incorporation was to bc investigated, namely phenylalanine, methionine, leucine, and tyrosine. When the labeled pulse medium was prepared for an experiment, all these constituents were replaced in the recommended amounts, and in the unlabeled form, except for the one amino acid which was to be studied and was in the labeled form. Tritiated amino acids were obtained from the Radiochemical Centre, Amersham, and their concentration in the experiments was kept as high as possible (100 &/ml) by using small volumes of pulse medium (1.0 ml). Prior to each experiment the fluke slices were rinsed in unlabeled Ml99 (at 37 C) which lacked the amino acid under study. Several slices were fixed in 10% formaldehyde in Millonig’s buffer pH 7.4, to act as unlabeled controls, and the remainder were placed for 5 min in the pulse chamber (Fig. l), with 1.0 ml of labeled medium. The temperature was maintained at 37 C by a heating jacket (J, Fig. 1) through which water was circulated, and a fine stream of gas (95% Na, 5% COz), was passed through the medium, providing gentle agitation and an anaerobic environment. After pulsing, the slices were rinsed in warm chase medium containing the test molecule in unlabeled form, and several slices were fixed immediately as for the controls. The remaining slices were incubated in chase medium, at 37 C under
AUTORADIOGRAPHY
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FIG. 1. Incubation chamber and connections. G, rubber hose to 95% N2/5% CO, gas supply; B, water-filled chamber through which gas is bubbled; H, hypodermic needles, V, vent for incubation chamber; I, incubation chamber surrounded by a jacket, J, through which warm water circulates in the direction of the white arrow.
anaerobic conditions with rocking, and several slices fixed after 5, 10, 20, 30, 45, and 60 min. All the slices were fixed for 24 hr at 4 C in 10% formaldehyde in Millonig’s buffer, pH 7.4. This fixative was used instead of glutaraldehyde because the latter binds unincorporated amino acids to structural proteins by cross-linking, and, therefore, gives fixation artifacts in autoradiograms (Hodgson and Marshall 1967; Peters and Ashley 1967, 1969), Formaldehyde is a satisfactory fixative for electron microscopy (Baker and McCrea 1966) and does not give this artifact (Peters and Ashley 1967, 1969). After fixation, the slices were washed in Millonig’s buffer, and post fixed in 1% osmium tetroxide in Millonig’s buffer, pH 7.4. The slices were then dehydrated by an ethylene glycol series, 20, 50, 75, and 90% for 30 min each, then 100% for 24 hr with frequent changes, passed through pro-
pylene oxide (4 hr) and embedded in Araldite. This procedure was adopted after several unsuccessful experiments had been carried out using alcohol as the dehydrant. It is possible that alcohol extracts considerable amounts of protein and glycoprotein from tissue fixed in formaldehyde and it is known that “inert” dehydrants, such as ethylene glycol, retain many low molecular weight substances in substantially unaltered quantities (Pease 1965; Sjostrand 1971). Preparation for electron microscope autoradiography. The techniques and problems of electron microscope autoradiography have been critically reviewed and discussed by many authors including Caro (1962, 1966), Caro and van Tubergen (1962), Granboulan ( 1965), Jacob ( 1971), Pelt ( 1963), Salema ( 1970), and Salpeter and Bachmann ( 1965). The method used in this investigation was based on the “loop”
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technique of Caro and van Tubergen ( 1962), which is one of the simplest techniques available and which has been used successfully in ‘a number of important studies (Caro and Palade 1964; Jamieson and Palade 1967b, 1968a, b; Shannon and Bogitsh 1971). UItrathin sections were cut from the slices embedded in Araldite using an LKB III ultramicrotome and mounted on Formvar-coated grids of nickel. The coating procedure was carried out under darkroom conditions using a Kodak minimum red safelight mounted about 3 ft from the working area. The emulsion solution was prepared in a Coplin jar by dissolving 10 g of Ilford L4 nuclear research emulsion (gel form) in 20 ml of distilled water at 45 C. The mixture was maintained at 45 C in a waterbath for 15 min and was frequently stirred with a paraffin coated glass rod, The emulsion solution was then immersed in an ice bath for 2 min, stirred, and kept at room temperature for a further 30 min, with occassional stirring, before use. For coating each grid was balanced (section side upward), on a wooden peg (3 mm diam), a number of which were mounted vertically on a baseboard. A film of gelled emulsion was formed across a Nichrome wire loop by dipping the latter into emulsion, withdrawing it gently, and holding it with the film vertical for 5 sec. The film was then applied to the grids by bringing the loop down briskly over the peg, at an angle of 45”, completely to the baseboard. The film generally burst within seconds leaving a thin layer of emulsion
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adhering to the surface of the grid and sections. After coating, each grid was stuck by its edge ‘and with the sections uppermost to a strip of double-sided sellotape mounted on a clean glass slide. Several grids were stuck to each slide and when two slides were completed, they were labeled appropriately on their free surfaces with a black wax pencil and placed in plastic two slide containers with the grids facing outward. The space between the slides was packed with silica-gel crystals and the container sealed and labeled. Each container had previously been made light-tight with black insulating tape ‘and enamel paint. The containers associated with each experiment were packed together in a light- and air-tight box, with silica-gel, and were stored at 4 C for 5 mo. After the exposure period, the grids were gently removed from the slides and developed at 20 C in Kodak D-19 developer (2 min). Following a rinse in 1% acetic acid, the preparation was fixed in Ilford “Hypam” fixer ( 1 in 10 dilution for 5 min), and washed for 30 min in distilled water which was changed frequently. Processing was carried out in the depressions of white porcelain tiles and in embryo dishes and it was found that cocktail sticks and slightly magnetic forceps were convenient for manipulating the grids. Prior to electron microscope examination, the autoradiograms were treated with an aqueous solution of 0.2% sodium hydroxide for 90 set, in order to remove the gelatin of the photographic emulsion. The sections were then stained for 5 min with
FIG. 2. i3Hltyrosine incubation; 5 min pulse; 5 min chase. Silver grains (white arrows) are present at the base of the gut epithelial cells (B) and the basal granular endoplasmic reticulum, (ER). Activity is also associated with muscle blocks (MU) in the thick layer of interstitial connective tissue (I). X25,000. FIG. 3. [“Hltyrosine; 5 min pulse, 5 min chase. Silver grains are present over the granular endoplasmic reticulum (ER), adjacent to the lateral plasma membranes of the cells (L). Note that the membranes of the ER cistemae appear as negative images (“white” lines), and the cisternal contents as a “dark” line. M, mitochondrion showing a negative image of its outer, inner, and qristal membranes. X40,000,
FIG. 4. [3H]tyrosine; 5 min pulse, 20 min chase. Grains are present in the vesicular component (GV) of the Golgi complex and in adjacent ER, condensing vacuoles (CV), and secretory bodies ( S) . M, mitochondrion. ~40,000. FIG. 5. [3H]tyrosine; 5 min pulse, 20 min chase. Silver grains are associated mainly with secretory bodies (S), but some also lie over the endoplasmic reticulum (ER). N, nucleus. x34000. 172
FIG. 6. [3H]tyrosine; 5 min pulse; 30 mm chase. The cell apex is shown and silver are associated with secretory bodies (S), Golgi vesicles (GV), and the apical lamellae which project from the cell surface into the lumen. ~25,000. FIG. 7. [‘Hltyrosine; 5 min pulse, 30 min chase. Grains overlie a condensing vacuole in the apical part of the cell. LA, lamellae. X65,000. FIG. 8. [$H]methionine; 5 min pulse, 30 mm chase. A silver grain lies over a secretory ( S ) in the process of being extruded into the lumen of the gut. LA, lamellae. X62,500. 173
grains (LA) (CV) body
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alcoholic uranyl acetate and for 4 min in lead citrate. The unincubated control material was coated with photographic emulsion and subsequently treated for autoradiography along with the incubated material. The sections of material from the controls showed no labeling over the gut cells. RESULTS
All four amino acid tracers ( [3H]tyrosine, [ 3H]methionine, [ 3H] leucine, and [ 3H]phenylalanine), were found to be incorporated by the epithelial cells of the gut and the distribution of the label within the cells varied with the length of the chase period. The structures of the cells over which silver grains occurred were the GER, the Golgi complexes, the secretory bodies, and the apical lamellae, and, regardless of chase period, labeled cells showed activity over most of these sites. However, the frequency with which grains occurred over the various sites was dependent on the length of the chase period. With material which had been given short chase periods (O-10 min), most of the silver grains were found over the GER, particularly that near the base of the cells and adjoining the lateral plasma membranes (Figs. 2 and 3). After a longer chase period (20 min), much of the activity was located at or near the Golgi complexes. Grains were observed over the GER adjoining the Golgi, over the small transition vesicles between the GER and the Golgi, and over the large secretory bodies arising from the maturing face of the Golgi complex (Figs. 4 and 5). In material which had been given 30 or 45 min chase periods, labeling was predominantly in the apical regions of the cells and was associated with
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the secretory bodies (Figs. 6 and 7)) with discharging secretory material at the cell surface (Fig. S), or between the lamellae (Fig. 9), and with the lamellae themselves (Fig. 10). More activity was present over the lamellae after 45 min chase than after 30 min, and one autoradiogram of the former material showed labeling associated with flocculant material in the gut lumen (Fig. 11). Little activity was detected in material which had received a 60-min chase period. In order to check that the localization of activity in labeled cells was influenced by the length of the chase period, the distribution of silver grains in a series of autoradiograms of [3H]methionine labeled slices was analysed. This was done by noting in each autoradiogram the ultrastructural localization of the silver grains. The labeled sites were classified into the following groups; GER, GER associated with the Golgi complex, Golgi complex, secretory bodies at the cell apex, and lamellae. For each chase period a total of 200-300 grains in two or three autoradiograms was recorded and the number of grains lying over each of the five ultrastructural categories expressed as a percentage of the total count (Fig. 12, Table 1). The results are illustrated in Fig. 12, which shows that immediately after labeling most activity is over the GER and that during the succeeding chase periods a “wave” of activity passed through the cells. The labeled material moved from the GER to the Golgi and eventually reached the apex of the cells in the secretory bodies. From the apex it was discharged and became associated with the lamellae. The movement was not fully synchronised, however, and a certain amount -
FIG. 9. [‘Hlphenylalanine; 5 min pulse, 45 min chase. A grain lies over a number of lamellae and near a secretory body (S), Th e 1amellae appear as dense cores of cytoplasm (C) flanked by negative images of membranes and an outer fuzzy coat. X120,000. FIG. 10. [“Hltyrosine; 5 min pulse, 30 min chase. Grains are associated with lamellae (LA) and flocculant material in the lumen (white arrow). X75,000. FIG. 11. [3H]tyrosine; 5 min pulse, 45 min chase. A silver grain (white arrow) lies over flocculant material in the gut lumen. X40,000.
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12 10
20
JO
45 min
FIG. 12. Incorporation of [‘Hlmethionine into gut epithelial cells. The graphs show the % of incorporation with time into different organelles and structures. ER, granular endoplasmic reticulum; ER/GC, boundary between the endoplasmic reticulum and the forming face of the Golgi complex; GC, maturing sacs of the Golgi complex; S, secretory bodies; LA, lamellae.
of activity was left behind at each ultrastructural location. TABLE Distribution [“HI
of Siluer Methionine Cells,
After
yO Total
Location
GER GEIt/Golgi Golgi Secretory bodies Lamellae --.--.-
I
Grains in Autoradiograms Pulse-La&led Epithclial Various Chase Periods
___ ~--
grains periods
of
after chase of ____
0 min
10 min
20 min
30 min
45 min
60 13 9
43 26 14
22 18 24
20 11 28
12 4 27
10 8
13 4
19 17
27 14
31 26
DISCUSSION
Protein synthesis by the epithelial cells. The results of the experiments with tritiated amino acids show clearly that the GER and Golgi complexes of the epithelial cells of the gut of Fasciola hepotica are involved in the synthesis of protrin-containing material, which is subsequently discharged into the gut lumen. The mechanism involved is similar to that which has been demonstrated by autoradiographic methods for vertebrate cells (Caro and Palade 1964; Jamieson and Palade 1967a; 1968a, b, c), and it agrees closely with
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the mechanism proposed by Robinson ( 1971)) on morphologic21 evidence. The common occurrence of activity in the GER near the base and lateral margins of the cells in material from short chase periods, suggests that the labeled amino acids entered across the basal and lateral membranes, rather than across the apical membrane. The interstitial material may have a role in bringing precursors within the range of the transport membranes since it occurs as a thick layer immediately beneath the basal lamina of the cells. On the other hand the inpushings of the parenchymal cells into the base of the gut epithelial cells may be a more important region of transfer and the junctional complexes between parenchymal and gut cells may be the sites of precursor transport and are known to contain acid and alkaline phosphatases (Threadgold 1968; Gallagher and Threadgold 1967; Robinson 1971) . This problem might be resolved by electron microscope autoradiography with frozen or freeze-dried sections. Since, after the shortest chase periods, most of the activity was confined to the GER, this region must be the first in which amino acids are incorporated into nondiffusable molecules. This is identical to the situation in vertebrate tissues (Caro and Palade 1964; Redman, Siekevitz, and Palade 1966), and suggests that the mechanism for protein synthesis which operates in F. hepatica is very similar to that in vertebrates, i.e., manufacture of polypeptide and protein chains at the ribosomal sites, with subsequent passage of the completed molecules into the GER cisternae for transport within the cell. The concentration of activity in the GER adjacent to Golgi complexes, after a lo-min chase, suggests that the labeled protein was transported to this region in the GER cisternae. Subsequently the activity appeared in the Golgi complexes themselves, and was associated with the small pe-
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ripheral vesicles which bud off from the GER (Robinson 1971) and with the large secretory vesicles. This suggests that the small vesicles (transition vesicles) are vehicles for the transfer of secretory proteins from the GER to the condensing vacuoles (secretory vesicles ) of the Golgi complexes. Thus these vesicular components are probably analogous to the vesicular structures present around and within the Golgi complexes of vertebrates (Jamieson and Palade 1967a, b). The labeled condensing vacuoles were found to concentrate toward the apices of the cells after long chase periods and formed into clusters. Discharge of these labeled secretory bodies was effected by eccrine secretion (Kurosumi 1961). In this process the limiting membrane of the secretory body fuses with the apical membrane of the cell, hence becoming part of the membrane of the lamellae, and the contents are released onto the surface. The frequent occurrence of activity on the lamellae of the epithelial cells after long chase periods suggests that some of the labeled amino acids were incorporated into, or intimately bound to the membranes of the secretory bodies, rather than with their contents. It is possible that the inner aspect of these membranes is lined with glycoproteins, which become part of the glycocalyx of the lamellae when the contents of the secretory bodies is discharged. Most of the activity associated with the discharged contents of the secretory bodies was probably lost during processing for electron microscopy, since this material, which is perhaps a proteolytic enzyme, would lie free in the gut lumen. The results show that the movement of radioactivity through the epithelial cells was not entirely synchronous and labeled material was retained at each stage of the synthetic process. This may have been partly due to the incorporation of label into structural protein or cytoplasmic enzymes. A similar phenomenon was ob-
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served in the goblet cells of the mouse colon by Rhor and Richter (1967), and these authors used the term “sessile protein” to describe the labeled material remaining in cells. The present work has shown that the GER and Golgi complexes in the epithelial cells of the gut of F. hepatica carry out protein or glycoprotein synthesis by a mechanism broadly similar to that which occurs in vertebrate cells. This provides a basis for further detailed studies on protein synthesis in flukes. In particular, the mctabolic requirements of the various stages in the synthetic process could be investigated and the “energy-locks” identified. Studies of this nature have been carried out on vertebrates by Jamieson and Paladc (1967a, b, 1968a, b, c), and a comparison with an invertebrate system would be of great interest. Comment on the clehydmlion technique. Ethylene glycol appeared to preserve many of the molecules which were lost when alcohol was used as the dehydrant, but the ultrastructural appearance of the tissues dehydrated with ethylene glycol was rather unusual. The membranes appeared as “negative images” and dense “lines” were often observed in the centre of GER cisternae. Such “lines” probably represented peptides and proteins which were lost from the cisternal lumen during alcohol dehydration. Large gaps and spaces were common in and between cells, and much of the conventional ultrastructural detail of nuclei and muscle tissue was missing. Sjostrand ( 1971)) found that ethylene glycol dehydration gave particularly good preservation of membranes, but his staining technique (phosphotungstic acid) was different from that used here. Hence it might be possible to improve the appearance of the tissues dehydrated through ethylene glycol by altering the staining procedure. The appearance of gaps and spaces in the tissues may have been due to severe
shrinkage of cells following the transfer to propylene oxide, and this could perhaps be remedied by increasing the concentration of propylene oxide in a gradual manner. With such technical modifications as these to improve the ultrastructural appearance of the tissues, the ethylene glycol dehydration technique should prove useful in further autoradiographic studies on F. lzepaticn and perhaps other helminths. REFERENCES BAKER, J. R., AND MCCREA, J. XI. 1966. The fine structure resulting from fixation by formaldehyde. The effects of concentration duration, and temperature. Journal of the Ro!yal Microscopical Society 85, 391-399. BJOHKMAN, N., AND THORWLL, W. 1964. On the fine structure of the cuticle of the liver fluke, Fasciola hepatica L. Experimental Cell Re.search 33, 319-329. BURTON, P. R. 1962. In zjitro uptake of radioglucose by a frog lung-fluke, and correlation with the histochemical identification of glycogen. Journal of Parasitology 48, 874882. CARO, L. G. 1962. High resolution autoradiography 11. The problem of resolution. Journal of Cell Biology 15, 189-199. CAIIO, L. G. 1966. Progress in high resolution autoradiography. Progress in Biophysics and Molecular Biology 16, 171-190. CARO, L. G., AND PALADE, G. E. 1964. Protein synthesis, storage, and discharge in the pancreatic exocrine cell. An autoradiographic study. Journal of Cell Biology 20, 473-496. CARP, L. G., AND VAN TUBERGEN, R. P. 1962. High Resolution autoradiography. I. Methods. Journal of Cell Biology 15, 173-188. ESWER, E. II., AND NOVIKOFF, A. B. 1962. Cytological studies on two functional hepatomes. Interrelationships of endoplasmic reticulum, Golgi apparatus, and lysosomes. Journal of Cell Biology 15, 189-312. GALLAGHER, S. S. E., AND THREADGOLD, L. T. 1967. Electron microscope studies of Fa.sciola hepatica. II. The interrelationship of the parenchyma with other organ systems. Parasitology 57, 627-632. GIIANBOULAN, P. 1965. Comparison of emulsion and techniques in electron microscope radioautography. Sympos!um of the International Society for Cell Biology 4, 43-63. IIANNA, K. E. B., AND THREAXOLII, L. T. 1975. Development of an in vitro technique for
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cytological investigations of slices of Fasciola hepatica: Evaluation by Morphological Criteria. International Journal for Parasitology, in press. IIO~GSON, S., AND MARSHALL, J. 1967. Tyrosine incorporation into the rabbit retina. Journal of Cell Biology 35, 722-726. JACOB, J. 1971. The practice and application of electron microscope autoradiography. International Review of Cytology 30, 91-181. JA~\IIESON, J. D., AND PALADE, G. E. 1967a. Intracellular transport of secretory proteins in the pancreatic exocrine cell. I. Role of the peripheral elements of the Golgi complex. Journal of Cell Biology 34, 577-595. JAMIESON, J. D., AND PALADE, G. E. 1967b. Intracellular transport of secretory proteins in the pancreatic exocrine cell. II. Transport of condensing vacuoles and zymogen granules. Journal of Cell Biology 34, 596-615. JAMIESON, J. D., AND PALADE, G. E. 1968a. Intracellular transport of secretory proteins in the pancreatic exocrine cell. III. Dissociation of intracellular transport from protein synthesis. Journal of Cell Biology 39, 580-587. JAAUESON, J. D., AND PALADE, G. E. 1968b. Intracellular transport of secretory proteins in the pancreatic exocrine cell. IV. Metabolic requirements. Journal of Cell Biology 39, 58% 603. JASIIESON, J. D., AND PALADE, G. E. 1968c. Metabolic requirements of zymogen discharge. Journal of Cell Biology 39, 66a. KUROSUMI, K. 1961. Electron microscope analysis of secretion. International Review of Cytology 11, l-124. LUMSDEN, R. D. 1966. Cytological studies on the absorptive surfaces of cestodes. II. The synthesis and intracellular transport of protein in the strobilar integument of Hymenolepis diminuta. Zeitschrift fur Parasitenkunde 28, l-13. NOLLEN, P. M. 1968. Uptake and incorporation of glucose, tyrosine, and thymidine by adult Philopthalmus megaluris (Lost 1914) (Trematoda), as determined by autoradiography. Journal of Parasitology 54, 295304. OAKES, J. A., AND LUMSDEN, R. D. 1971. Cytological studies on the absorptive surfaces of cestodes. V. Incorporation of carbohydratecontaining macromolecules into tegment membranes. Journal of Parasitology 57, 12561268. PAPPAS, P. W. 1971. Haematoloechus medioplexus: uptake, localization, and fate of tritiated arginine. Experimental Parasitology 30, 102-119.
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PEASE, D. C. 1965. Polysaccharide associated with the exterior surface of epithelial cells: kidney, intestine, brain. Journal of Ultrastructural Research 15, 555-588. PELC, S. R. 1963. Theory of electron autoradiography. Journal of the Royal Microscopical Society 81, 131-139. PETERS, T., AND ASHLEY, C. A. 1967. An artifact in radioautography due to the binding of free amino acids to tissues by fixatives. Journal of Cell Biology 33, 53-60. PETERS, T., AND ASHLEY, C. A. 1969. In “Autoradiography of Diffusible Substances (L. J. Roth and W. E. Stumpf, eds.), pp. 267-278. Academic Press, New York. REDILIAN, C. M., SIEKEVITZ, P., AND PALADE, G. E. 1966. Synthesis and transport of amylase in pigeon pancreatic microsomes. Journal of Biological Chemistry 241, 1150-1158. ROBINSON, G. 1971. “Experimental Studies on the Common Liver Fluke, Fasciola hepatica L.” Ph.D. Thesis, Queen’s University, Belfast. RHOR, H., AND RICHTER, H. 1967. Vergleichende elektronenmikroskopisch - autoradiographische Untersuchungen uber den eiweissstoffwechsel der Zelle Unter besonderer Berucksichtigung der Colonbecherzelle. Patholgia Europaea 2, 280-301. SALEMA, R. 1970. Autoradiography with the electron microscope. Principals and Techniques. Broteria Serie trimestral: Ciencias Natwais 39, 109-129. SALPETER, M. M., AND BACHMANN, L. 1965. Assessment of technical steps in electron microscope autoradiography. Symposium of the International Society for Cell Biology 4, 2341. SHANNON, W. A., AND BOGITSH, B. J. 1971. Megalodiscus temperatus: Comparative radioautography of glucose H3 and galactose H8 incorporation. Experimental Parasitology 29, 309-319. SIEKEVITZ, P. 1959. The cytological basis of protein synthesis. Experimental Cell Research Supplement 7, 90-110. SJOSTRAND, F. S. 1971. Molecular structure and function of cellular membranes. In “Cell Membranes; Biological and Pathological Aspects,” pp. l-29. The American Association of Pathologists and Bacteriologists. THORSELL, W., APPELCREN, L. E., AND KIPPAR, M. 1968. Distribution and fate of 2-C’4-glucose in the liver fluke, Fasciola hepatica L. after short in vitro incubation. Zeitschrift fur Parasitenkunde 31, 113-121. THORSELL, W., BJORKMAN, N., AND APPELGREN, L. E. 1966. Radioautographic studies on the ovary and vitelline glands of Fmciola hepatica
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after short in citro incubation with some amino acids. ZeitCdzrift fur Parasitenkunde 28, 108-115. THREADGOLD, L. T. 1968. Electron microscope studies of Fasciola hepatica. VI. The ultrastructural localization of phosphatases. Erperimental Parasitology 23, 264-276.
THREADGOLD,
L. T.,
Development cytological hepatica: teria.
AXI)
of Evaluation
in press,
in
investigations
International
R. E. IS. 1975.
IIASNA,
an
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technique
of slices by
Journal
Physiological for
fol
of Fasciola Parasitology,
Cri-