- Email: [email protected]
Fasciola hepatica: A light and electron autoradiographic study of shell-protein and glycogen synthesis by vitelline follicles in tissue slices
EXPERIMENTAL
PARASITOLOGY
39,
18-28 (1976)
Fasciola hepatica: A Light and Electron Autoradiographic Study of Shell-Protein and Glycogen Synthesis by Vitelline Follicles in Tissue Slices R. E. B. HANNA l IPresent address: E.A.V.R.O., Zoology Department, (Accepted
Queeni
Muguga,
P.O. Box 32, Kiiyu,
University,
for publication
Kenya.
Belfast, North Ireland
February 27, 1975)
IIANNA, Ft. E. B. 1976. Fusciok hepdica: A light and electron autoradiographic study of shell-protein and glycogen synthesis by vitelline follicles in tissue slices. Experimental Parazitology 39, 18-23. The incorporation of tritiated amino acids and monosaccharides by the vitelline cells of F. hep&x slices maintained in vftro was studied by light and electron microscope autoradiography. A “pulse-chase” labeling technique was used with tritiated tyrosine, phenylalanine, leucine, and methionine, of which Hs-tyrosine was the most readily incorporated into shell-protein globules of immature vitelline cells. The mechanism of protein synthesis appeared to resemble the GER-Golgi mediated mechanism of vertebrates. Young vitelline cells were the most active in protein synthesis, and they matured considerably during the 60 min chase period. Maturing cells, which were carrying out glycogenesis, incorporated no amino acids. An “accumulation” labeling technique was used with Hs-galactose and Hsglucose. Both monosaccharides were readily incorporated into glycogen by vitelline cells which had reached the stage of glycogenesis, but mature cells, which were already packed with glycogen, incorporated little monosaccharide. Labeling appeared in the nurse cells of follicles containing many mature vitelline cells. No evidence was found for the involvement of any cell organelle in glycogenesis, but preformed glycogen may have acted as a “template” for further synthesis. INDEX DEXXUFTORS: Faxiola hepatica; Vitellaria; Nurse cells; Shell-protein synthesis; Glycogenesis; Tyrosine; Phenylalanine; Leucine; Methionine; Galactose; Glucose; Autoradiographic light and electron microscopy.
in them during development, have been described in detail by Irwin (1970) and Irwin and Threadgold ( 1970). These authors showed that the vitelline follicles contain clusters of cells in various stages of cytomorphosis. Immature cells have a typical “embryonic” morphology, and are found peripherally, where they are continuously produced by mitotic division. Young vitelline cells soon develop an extensive GER system, and Golgi bodies appear which give rise to globules of shell protein. These globules migrate to the periphery of each cell and aggregate there
INTR~Du(;TIoN
Electron microscope autoradiographic on slices of Fasciolu hepatica maintained in vitro have shown that the gut cells of this fluke carry out synthesis and secretion of protein-containing material, by a mechanism similar to that which operates in vertebrate tissues (Hanna 1975). In the present report, the results of autoradiographic studies on protein and glycogen synthesis of the vitelline cells of similar in vitro preparations will be presented. The ultrastructure of the vitelline cells, and the morphological changes which occur studies
18
COpyrieht 1976 by Academic Press, All rights oQ reproduction in any form
Inc. reserved.
AUTORADIOGRAPHY
IN
F.
hf3fXZtiCU
TABLE Summary Tracer
Amino acids L-tyrosine-3,VH L-3-phenylalanine L-leucine-4,5-3H L-methionine D-galactose-1-3H
D-glucose-2JH
Specific activity (Ci/mmole)
(ring-4-3H) (methyl-3H)
25 20 1.0 7.5
Concentration in labeling medium W&W
18.4
500
0.5
83
in membrane-bound clusters. Later in the life of the cell, when several vitelline cells associate with an ovum in the iiotype, the shell-protein globules are released, and form a layer of eggshell around the cell mass. As maturation continues in the vitelline follicle, protein production ceases, the GER and the cell converts to degenerates, glycogen synthesis. The space between the pyknotic nucleus and the peripheral protein globules becomes filled with glycogen and yolk bodies (membrane-sequestered areas of glycogen and GER, which are probably autolysosomes) . Mature cells, which have reached the center of the follicle, break away and move down the vitelline ducts. Nurse cells are also present in the follicles, and cytoplasmic processes from these completely envelop the growing vitelline cells. The nurse cells may select and transport precursors for protein and glycogen synthesis in the vitelline cells. The researches outlined above suggest that experimental studies using radioactive tracer molecules might profitably be used to elucidate further the processes involved in shell-protein synthesis and glycogenesis,
19
CELLS
I
of Experimental
100
VITELLINE
Dkgn Slices
Labeling technique used
prepared
for:
Pulse-chase 5 min pulse; 0, 5, 10, 20, 30, 45, 60 min chase
L.M. autoradiography EM. autoradiography (ethylene glycol dehydration)
Accumulation 5, 10, 20, 30, 45, 60 min
L.M.arg. and E.M. (alcohol dehydr.)
Accumulation 5, 20 min
EM. are. dehydi.)
and these research.
are the aims
MATERLALS
AND
and
arg.
(alcohol ‘onl)
of the
present
METHODS
a. Tissue preparation media and in vitro conditions. Adult flukes from infected rats were collected and sliced as described previously ( Hanna and Threadgold 1975), and labeling was carried out in the apparatus described by Hanna ( 1975). The medium used was Ml99 (bicarbonate buffered to pH 7.5 at 37 C; 290 m OsM ) with antibiotics (sodium benzylpenicillin, 1000 iu/ml; streptomycin sulphate, 2 mg/ml). A temperature of 37 C was maintained throughout incubation. A fine stream of gas (95% N%, 5% COB) was continuously passed through the incubation chambers, providing agitation and an anaerobic environment. All the precursors used (tyrosine, phenylalanine, leucine, methionine, galactose, and glucose) were lat,sled with tritium, and were obtained fron, the Radiochemical Centre, Amersham. The;v concentrations in the experiments were k,xpt as high as possible by using small volumes of incubation medium (1 ml).
20
R. E. B. HANNA
AUTORADIOGRAPHY
IN
F.
The method of preparing the radioactive and chase media containing amino acids was that previously described (Hanna 1975). A similar method was adopted for H3-glucose and H3-galactose, the medium being free of other sugars when either of these was in the medium. The specific activity and concentration of the different labels are given in Table I. Prior to labeling, the slices were rinsed in unlabeled Ml99 which lacked the precursor whose incorporation was to be examined; and in those experiments which involved a “chase” incubation, after a radioactive “pulse,” the chase medium was normal M199, with the test molecule present in unlabeled form. 12. Labeling techniques. With the amino acids, a pulse-chase technique was used for labeling the slices. This method has been fully described elsewhere (Hanna 1975). After a pulse incubation of 5 min in labeled medium, the slices were rinsed, and then incubated for periods of 0, 5, 10, 20, 30, 45, and 60 min in chase medium. A few slices were fixed without “pulse” incubation, to provide unlabeled controls. After each chase period, several slices were fixed in 10% formaldehyde in Millonig’s buffer, pH 7.4. Fixation was continued for 24 hr at 4 C, and was followed by washing in several changes of Millonig’s buffer. Several slices from each chase period were postfixed with 1% Millonig
he@iCU
VITELLINE
CELLS
21
buffered osmium tetroxide, pH 7.4, dehydrated in an ethylene glycol series, brought through propylene oxide to Araldite, and embedded for electron microscopy (Hanna 1975). The remaining slices from each chase period were dehydrated in an ethylene glycol series and embedded directly in glycol methacrylate (GMA), for light microscopy. An “accumulation” technique was used for labeling the slices with the monosaccharide tracers, H”-galactose and H3-glucase because no movement of the tracer after its incorporation into glycogen in the cytoplasm of the cell was anticipated. In the galactose experiment, slices were incubated in the presence of radioactive precursor for up to 60 min, several being removed, rinsed, and fixed in 10% in formaldehyde after 5, 10, 20, 30, and 45 min. Several slices were fixed without prior incubation to provide unlabeled controls. All the slices were fixed for 24 hr and washed in Millonig’s buffer. Then several slices from each incubation time were postfixed in 1% Millonig buffered osmium tetroxide pH 7.4, dehydrated in an alcohol series, passed through propylene oxide, and embedded in Araldite for electron microscopy. The remainder were dehydrated in GMA for light microscopy. In the glucose experiment, only two incubation times were used (5 and 20 min) ; and all the slices were postfixed, dehy-
FIG. 1. Vitellaria. H:‘-tyrosine; 5 min pulse, 5 min chase. The immature cells (i) have many silver grains over their cytoplasm. Maturing cells (m) have grains over their cytoplasm and around their shell globules and clusters (S and arrows), whereas mature cells (fm) do not have grains around or over their shell globules (S ). x4500. Unstained, phase contrast. FIG. 2. Vitellaria. H3-tyrosine; 5 min pulse, 60 min chase. The imnature cells (i) are free of grains, whereas maturing (m) and some fully mature (fm) cells have many grains associated with their shell-protein globules and clusters. Some fully mature cells (fml) do not have grains. x5000. Unstained phase contrast. FIG. 3. Vitellaria. Ethylene glycol dehydration H”-phenylalanine; 5 min pulse, 0 min chase. A silver grain (arrow) is associated with the nurse cell cytoplasm, but not with the granular endoplasmic reticulum (ER) or shell globules (S ) of the adjacent vitelline cell. X50,000. FIGS. 4, 5. Vitellaria. Ethylene glycol dehydration. H3-tyrosine; 5 min pulse, 10 min chase. Grains (arrows) lie over the granular endoplasmic reticulum (ER, Fig. 4) and the Golgi region (GC, Fig. 5) and its condensing vacuoles (V). Grains are absent over shell globules (S). Fig. 4, x37,500; Fig. 5, X50,000.
22
R. E. B. HANNA
AUTORADIOGRAPHY
IN
F.
hf?patiCa
drated in alcohol, and embedded in Araldite for electron microscopy. No light microscope autoradiograms were prepared. The important features of the experimental design are summarized by Table I. c. Preparation of light microscope autoradiograms. lprn sections of the slices embedded in GMA were cut with glass knives using a Reichert Om U2 ultramicrotome. Several sections from each block were mounted, in a drop of water, near the end of an appropriately labeled microscope slide, and when all the slides were dry, they were dipped, section ends downward in a solution of 10 g of Word L4 nuclear research emulsion in 20 ml of distilled water at 45 C. Excess emulsion was allowed to drain from the slides by standing them on absorbent paper for 10 min. Then the preparations were hung in a warm dustfree atmosphere for an hour to dry, after which they were packed in air- and lighttight boxes, with silica gel present as dessicant, and were stored at 4 C for a 5-6 wk exposure period. Coating, drying, and packing of the slides was carried out under darkroom conditions using a Kodak minimum red safelight, mounted 3 ft from the working area. After exposure, the autoradiograms were developed using Kodak D-19 developer (5 min at 20 C ) , washed with 1% acetic acid, and fixed using Ilford “Hypam” fixer (1 in 10 dilution for 5 min). The slides were examined unstained, or lightly stained with 1% toluidine blue (30 set at room temperature), using a Leitz orthomat microscope and camera attachment with phase contrast illumination. Micrographs were taken at a magnification of x 900 using Ilford Pan F black and
FIGS. 6, 7, 8, 9. Vitellaria. Ethylene glycol 5 min pulse, 20 min chase; Fig. 8: H”-leucine; occur over granular endoplasmic reticulum (El%, associated with condensing vacuoles (V) of the or over shell globules (S, Fig 9). Figs. 6, 7, and
VITELLINJZ
CELLS
23
white film, or Agfachrome 50L professional color film. d. Preparation of electron-microscope autoradiograms. The method used in this investigation was based on the “loop” technique of Caro and Van Tubergen ( 1962), which is one of the simplest techniques available and has been used successfully in a number of important studies (Caro and Palade 1964, Shannon and Bogitsh 1971). Details of the technique are given in Hanna (1975) and, therefore, will not be given here. RESULTS
a. Incorporation of amino acids. Light microscope autoradiograms of slices pulselabeled with H3-tyrosine showed that this precursor had been readily incorporated by developing vitelline cells. The silver grains were concentrated around and over the shell-protein globules, which were normally at the periphery of the cells. H3-phenylalanine and H3-leucine had also been incorporated by vitelline cells, but labeling was not as heavy as it was with H3tyrosine. No activity was detected in the vitelline cells of slices labeled with H3methionine. In material from short chase periods (O-20 min), the immature vitelline cells and those in which shell-protein globules had just begun to appear had many silver grains over their cytoplasm. More mature cells also had grains around their shell globules, but fully mature cells with large glycogen stores had no grains (Fig. 1). After 45 and 60 min chase periods, however, the immature cells and those in the early stages of shell-protein synthesis had no or very few grains, whereas maturing cells had very many grains around and
Figs. 6, dehydration. 5 min pulse, 20 min Figs. 6 and 9), nurse Golgi complex (Figs. 8, x50,000; Fig. 9,
7, and 9: H3-tyrosine; chase. Grains (arrows) cell cytoplasm (Fig. 7), 8 and 9) and close to ~20,000.
24
R.
E.
B.
HANNA
AUTORADIOGRAPHY
IN
F.
over their shell globules; fully mature cells had few or no grains (Fig. 2). Electron microscope autoradiograms of slices pulse-labeled with HVyrosine, H3phenylalanine, and H3-leucine (but not those labeled with H3-methionine), showed labeling of the vitelline cells, approximately two to five silver grains per cell. In material removed immediately after the 5 min pulse, silver grains were present in the cytoplasm of nurse cells and the granular endoplasmic reticulum (GER) of vitelline cells, but not elsewhere in the latter (Fig. 3). After a 10 min chase, grains occurred over the nurse cell cytoplasm, and over the GER and condensing vacuoles of the Golgi complex of vitelline cells (Figs. 4 and 5) ; grains were not present over shell-protein globules. After 20 min, grains were present over all the sites listed previously (Figs. 6-g)) but also lay over shellprotein globules and clusters (Fig. 10). After 30 and 45 min chase periods, many grains occurred over shell-protein globules and clusters (Figs. 11 and 12), but were rare over GER and Golgi complexes. Silver grains were never found over glycogen areas or nuclei, and control tissue was always free of autoradiographic reaction. b. Incorporation of monosaccharides. ,Only those vitelline cells in which glycogenesis was occurring were labeled by H3galactose and H3-glucose. The immature cells, which were still synthesizing protein
hepaticu
VITELLINE
CELLS
25
globules, had no activity associated with them (Figs. 13 and 15). Furthermore, the nuclei, GER, and protein clusters of labeled cells were generally free of autoradiographic reaction, all the activity being present in the glycogen storage areas and yolk bodies (Figs. 13-16). The maturing cells, even those within a single follicle, showed great variation in the amount of “H-galactose incorporation (Fig. 13). The most mature cells, which were packed with glycogen, had few silver grains associated with them and in follicles where many of the cells had reached this stage, the regions between the vitelline cells (nurse cell protrusions) had many grains in their cytoplasm (Fig. 14). The distribution of labeling in the vitelline follicles did not vary with the length of time for which the slices had been exposed to the monosaccharide precursors, but the density of Iabeling over the glycogen regions was greater in material which had received longer incubations. As in the amino acid experiments, control material, which had not been exposed to radioactive precursors, showed no autoradiographic reaction in the vitelline cells. DISCUSSION
a. Shell-protein synthesis. Of the three tritiated amino acids incorporated into shell protein by immature vitelline cells,
FIG. 10. Vitellaria. Ethylene glycol dehydration, H3-tyrosine; 5 min pulse, 20 min chase. Grains (arrows) are next to or over shell globules (S ). X50,000. Fm. 11. Vitellaria. Ethylene glycol dehydration. H3-phenylalanine; 5 min pulse, 30 mm chase. Two grains (arrows) are over the shelLglobule cluster (S ). ~50,000. FIG. 12. Vitellaria. Ethylene glycol dehydration. H3-tyrosine; 5 min pulse, 45 min chase. Many silver grams (arrows) are over the shell-globule cluster (S ). X50,000. FIG. 13. Vitellaria. Ha-galactose, 5 min accumulation. The vitelline cells have incorporated different amounts of label during the short incubation. Maturing cells (m), which are carrying out glycogenesis, are heavily labeled, whereas immature cells (i) have no activity and mature cells (ma), in which glycogen synthesis is almost complete are lightly labeled. s, unlabeled shell protein. Toluidine blue, phase contrast. X3300. FIG. 14. Vitellaria. H3-galactose, 60 min accumulation. Most vitelline cells in this follicle were nearly mature (ma) when incubated and show little activity. Maturing cells (m ), however, are heavily labeled and silver grains also occur in the nurse cell cytoplasm (arrows) between the nature cells. Toluidine blue, phase contrast. X28,000.
26
R. E. B. HANNA
AUTORADIOGRAPHY
IN
F. hepaticu
H3-tyrosine was the most readily used. This finding agrees with the reports of Thorsell, Bjorkman, and Appelgren (1966) on F. hepatica and Nollen (1968) on Philophthalmus megabrus, and is not surprising in view of the fact that di-tyrosine links are partly responsible for the cross linking, and hence the resistance, of F. heputica egg shells ( Ramalingam 1973). In electron micrograph autoradiograms, silver grains appeared in the following order with time: nurse cell cytoplasm, GER, and condensing vacuoles of the Golgi complex of vitelline cells, and finally shell protein globules and clusters. With long chase periods, especially the 45 min sample, the number of grains over shell protein globules and clusters was much greater than at earlier times. This sequence in the appearance of grains with time strongly suggests that the route of shell protein synthesis and formation of shell globules is similar to that which occurs in gut cell of this fluke (Hanna 1975)) and in vertebrate tissues (Caro and Palade 1964). This study, therefore, lends support to the suggestions of Irwin (1970), and Irwin and Threadgold ( 1970) that shell protein is synthesized by the GER; is transferred to the Golgi complex for condensation; and migrates, in membrane-bound packages, to the cell periphery. Clusters of shell globules accumulate in this region, each cluster being held intact by an enveloping membrane. The activity detected in the nurse cells was probably due to incorporation of labeled molecules into structural proteins or cytoplasmic enzymes. If precursors pass through these cells prior to entering the vitelline cells they probably do so in a
FIG. 15. Vitellaria. storage areas ( G and immature cells (i) are them and the parenchyma x8000. FIG. 16. Vitellaria. glycogen stores (G and plasmic reticulum (ER)
VITELLINJI
27
CELLS
soluble form, and so would not be detectable by the present techniques. Light microscope autoradiography showed that many of the labeled cells from long chase periods were more mature than those from short chase periods. This indicates that the young cells were the most active in protein synthesis, and that during the 60 min chase period these cells became considerably more mature, and produced large amounts of shell protein. b. Glycogen synthesis. H3-galactose and H3-glucose were both incorporated readily into those vitelline cells which were producing glycogen. The variations noted in the density of labeling, between different cells in the same follicle, were probably due to variations in the rate of glycogen synthesis. The precursors were most readily incorporated by those cells in the early stages of glycogen production, while mature cells, which were already packed with glycogen stores, incorporated little, if any. The H3-galactose labeling in nurse cell regions of follicles containing many mature vitelline cells may have been due to the deposition of glycogen, using precursors which had been intended for the vitelline cells but were no longer needed by these due to the cessation of glycogenesis. No matter which incubation periods were used, H3-glucose labeling occurred throughout the glycogen regions of the cells incorporating these precursors, and some activity was also present in degenerating GER in the yolk bodies. This supports the view that glycogen deposition occurs mainly in the proximity of preformed glycogen, which might act as
Hs-galactose, 45 min accumulation. Silver grams lie over glycogen arrow) in mature vitelline cells with shell-globule clusters (S ). The unlabeled but label (arrow) appears in the nurse cell (N) between (P). m is a maturing cell which has not yet started glycogenesis. 10 min chase. The label Ha-glucose, 20 min pulse, arrows) in the vitelline cell. No activity is present in or shell globules ( S ). ~20,800.
is confined to granular endo-
28
R. E.
B. HANNA
a “template” for further synthesis (Coimbra and Leblond ( 1966) for rat liver). Although the onset of glycogen synthesis in the vitelline cells is preceded by degeneration of the GER and is accompanied by growth (both of which allow space for glycogen storage), no evidence was found for the dirct participation of any cell organelle in this process. It therefore appears that glycogenesis is mediated by cytoplasmic enzymes in these cells. The ability of F. hepatica to use galactose in glycogen synthesis implies that the glycogenic enzymes are not specific for glucose or that galactose can be converted to glucose in this animal. It is the author’s intention to discuss this phenomenon in detail in a subsequent paper. REFERENCES 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. CARO, L. G., AND VAN TUBERGEN, R. P. 1962. High resolution autoradiography. I. Methods. Journal of Cell Biology 15, 173-188. COIMBRA, A., AND LEBLOND, C. P. 1966. Sites of glycogen synthesis in rat liver cells as shown by electron microscope radioautography after administration of glucose-Hs. Journal of Cell Biology 30, 151-175. HANNA, R. E. B. 1975. Fasciola hepatica: An
electron microscope autoradiographic study of protein synthesis and secretion in gut cells in tissue slices. Experimental Parasitology, in press. HANNA, R. E. B., AND THREADGOLD, L. T. 1975. Development of an in vitro technique for cytological investigations of slices of Fasciola hepatica: Evaluation by morphological criteria. International Journal for Parasitology, in press. IRWIN, S. W. B. 1979. Some aspects of egg production in Fasciola hepatica. Ph.D. Thesis, The Queen’s University, Belfast. IRWIN, S. W. B., AND THREADGOLD, L. T. 1970. Electron microscope studies on Fasciola hepatica. VIII. The development of the vitelline cells. Experimental Parasitology 28, 399411. NOLLEN, P. M. 1968. Uptake and incorporation of glucose, tyrosine, leucine, and thymidine by adult Philophthalmus megabrus (Lost 1914) (Trematoda), as determined by autoradiography. Journal of Pamsitology 54, 295304. RAMALINGAM, K. 1973. The chemical nature of the egg-shell of the liver fluke, Fasciola
hepatica. ology SHANNON,
International
3, 67-75. W. A.,
Megalodiscw
Journal
for Pam&-
AND
BOGITSH, B. J. 1971. Comparative radioof glucose Ha and galactose Hx
temperatus.
autography incorporation. Experimental Parasitology 29, 369319. THORSELL, W., BJORKMAN, N., AND APPELGREN, L. E. 1966. Radioautographic studies on the ovary and vitelline glands of Fasciola hepatica after short in vitro incubation with some amino acids. Zeitschrift fur Parasitenkunde 28, 108-115.
PARASITOLOGY
39,
18-28 (1976)
Fasciola hepatica: A Light and Electron Autoradiographic Study of Shell-Protein and Glycogen Synthesis by Vitelline Follicles in Tissue Slices R. E. B. HANNA l IPresent address: E.A.V.R.O., Zoology Department, (Accepted
Queeni
Muguga,
P.O. Box 32, Kiiyu,
University,
for publication
Kenya.
Belfast, North Ireland
February 27, 1975)
IIANNA, Ft. E. B. 1976. Fusciok hepdica: A light and electron autoradiographic study of shell-protein and glycogen synthesis by vitelline follicles in tissue slices. Experimental Parazitology 39, 18-23. The incorporation of tritiated amino acids and monosaccharides by the vitelline cells of F. hep&x slices maintained in vftro was studied by light and electron microscope autoradiography. A “pulse-chase” labeling technique was used with tritiated tyrosine, phenylalanine, leucine, and methionine, of which Hs-tyrosine was the most readily incorporated into shell-protein globules of immature vitelline cells. The mechanism of protein synthesis appeared to resemble the GER-Golgi mediated mechanism of vertebrates. Young vitelline cells were the most active in protein synthesis, and they matured considerably during the 60 min chase period. Maturing cells, which were carrying out glycogenesis, incorporated no amino acids. An “accumulation” labeling technique was used with Hs-galactose and Hsglucose. Both monosaccharides were readily incorporated into glycogen by vitelline cells which had reached the stage of glycogenesis, but mature cells, which were already packed with glycogen, incorporated little monosaccharide. Labeling appeared in the nurse cells of follicles containing many mature vitelline cells. No evidence was found for the involvement of any cell organelle in glycogenesis, but preformed glycogen may have acted as a “template” for further synthesis. INDEX DEXXUFTORS: Faxiola hepatica; Vitellaria; Nurse cells; Shell-protein synthesis; Glycogenesis; Tyrosine; Phenylalanine; Leucine; Methionine; Galactose; Glucose; Autoradiographic light and electron microscopy.
in them during development, have been described in detail by Irwin (1970) and Irwin and Threadgold ( 1970). These authors showed that the vitelline follicles contain clusters of cells in various stages of cytomorphosis. Immature cells have a typical “embryonic” morphology, and are found peripherally, where they are continuously produced by mitotic division. Young vitelline cells soon develop an extensive GER system, and Golgi bodies appear which give rise to globules of shell protein. These globules migrate to the periphery of each cell and aggregate there
INTR~Du(;TIoN
Electron microscope autoradiographic on slices of Fasciolu hepatica maintained in vitro have shown that the gut cells of this fluke carry out synthesis and secretion of protein-containing material, by a mechanism similar to that which operates in vertebrate tissues (Hanna 1975). In the present report, the results of autoradiographic studies on protein and glycogen synthesis of the vitelline cells of similar in vitro preparations will be presented. The ultrastructure of the vitelline cells, and the morphological changes which occur studies
18
COpyrieht 1976 by Academic Press, All rights oQ reproduction in any form
Inc. reserved.
AUTORADIOGRAPHY
IN
F.
hf3fXZtiCU
TABLE Summary Tracer
Amino acids L-tyrosine-3,VH L-3-phenylalanine L-leucine-4,5-3H L-methionine D-galactose-1-3H
D-glucose-2JH
Specific activity (Ci/mmole)
(ring-4-3H) (methyl-3H)
25 20 1.0 7.5
Concentration in labeling medium W&W
18.4
500
0.5
83
in membrane-bound clusters. Later in the life of the cell, when several vitelline cells associate with an ovum in the iiotype, the shell-protein globules are released, and form a layer of eggshell around the cell mass. As maturation continues in the vitelline follicle, protein production ceases, the GER and the cell converts to degenerates, glycogen synthesis. The space between the pyknotic nucleus and the peripheral protein globules becomes filled with glycogen and yolk bodies (membrane-sequestered areas of glycogen and GER, which are probably autolysosomes) . Mature cells, which have reached the center of the follicle, break away and move down the vitelline ducts. Nurse cells are also present in the follicles, and cytoplasmic processes from these completely envelop the growing vitelline cells. The nurse cells may select and transport precursors for protein and glycogen synthesis in the vitelline cells. The researches outlined above suggest that experimental studies using radioactive tracer molecules might profitably be used to elucidate further the processes involved in shell-protein synthesis and glycogenesis,
19
CELLS
I
of Experimental
100
VITELLINE
Dkgn Slices
Labeling technique used
prepared
for:
Pulse-chase 5 min pulse; 0, 5, 10, 20, 30, 45, 60 min chase
L.M. autoradiography EM. autoradiography (ethylene glycol dehydration)
Accumulation 5, 10, 20, 30, 45, 60 min
L.M.arg. and E.M. (alcohol dehydr.)
Accumulation 5, 20 min
EM. are. dehydi.)
and these research.
are the aims
MATERLALS
AND
and
arg.
(alcohol ‘onl)
of the
present
METHODS
a. Tissue preparation media and in vitro conditions. Adult flukes from infected rats were collected and sliced as described previously ( Hanna and Threadgold 1975), and labeling was carried out in the apparatus described by Hanna ( 1975). The medium used was Ml99 (bicarbonate buffered to pH 7.5 at 37 C; 290 m OsM ) with antibiotics (sodium benzylpenicillin, 1000 iu/ml; streptomycin sulphate, 2 mg/ml). A temperature of 37 C was maintained throughout incubation. A fine stream of gas (95% N%, 5% COB) was continuously passed through the incubation chambers, providing agitation and an anaerobic environment. All the precursors used (tyrosine, phenylalanine, leucine, methionine, galactose, and glucose) were lat,sled with tritium, and were obtained fron, the Radiochemical Centre, Amersham. The;v concentrations in the experiments were k,xpt as high as possible by using small volumes of incubation medium (1 ml).
20
R. E. B. HANNA
AUTORADIOGRAPHY
IN
F.
The method of preparing the radioactive and chase media containing amino acids was that previously described (Hanna 1975). A similar method was adopted for H3-glucose and H3-galactose, the medium being free of other sugars when either of these was in the medium. The specific activity and concentration of the different labels are given in Table I. Prior to labeling, the slices were rinsed in unlabeled Ml99 which lacked the precursor whose incorporation was to be examined; and in those experiments which involved a “chase” incubation, after a radioactive “pulse,” the chase medium was normal M199, with the test molecule present in unlabeled form. 12. Labeling techniques. With the amino acids, a pulse-chase technique was used for labeling the slices. This method has been fully described elsewhere (Hanna 1975). After a pulse incubation of 5 min in labeled medium, the slices were rinsed, and then incubated for periods of 0, 5, 10, 20, 30, 45, and 60 min in chase medium. A few slices were fixed without “pulse” incubation, to provide unlabeled controls. After each chase period, several slices were fixed in 10% formaldehyde in Millonig’s buffer, pH 7.4. Fixation was continued for 24 hr at 4 C, and was followed by washing in several changes of Millonig’s buffer. Several slices from each chase period were postfixed with 1% Millonig
he@iCU
VITELLINE
CELLS
21
buffered osmium tetroxide, pH 7.4, dehydrated in an ethylene glycol series, brought through propylene oxide to Araldite, and embedded for electron microscopy (Hanna 1975). The remaining slices from each chase period were dehydrated in an ethylene glycol series and embedded directly in glycol methacrylate (GMA), for light microscopy. An “accumulation” technique was used for labeling the slices with the monosaccharide tracers, H”-galactose and H3-glucase because no movement of the tracer after its incorporation into glycogen in the cytoplasm of the cell was anticipated. In the galactose experiment, slices were incubated in the presence of radioactive precursor for up to 60 min, several being removed, rinsed, and fixed in 10% in formaldehyde after 5, 10, 20, 30, and 45 min. Several slices were fixed without prior incubation to provide unlabeled controls. All the slices were fixed for 24 hr and washed in Millonig’s buffer. Then several slices from each incubation time were postfixed in 1% Millonig buffered osmium tetroxide pH 7.4, dehydrated in an alcohol series, passed through propylene oxide, and embedded in Araldite for electron microscopy. The remainder were dehydrated in GMA for light microscopy. In the glucose experiment, only two incubation times were used (5 and 20 min) ; and all the slices were postfixed, dehy-
FIG. 1. Vitellaria. H:‘-tyrosine; 5 min pulse, 5 min chase. The immature cells (i) have many silver grains over their cytoplasm. Maturing cells (m) have grains over their cytoplasm and around their shell globules and clusters (S and arrows), whereas mature cells (fm) do not have grains around or over their shell globules (S ). x4500. Unstained, phase contrast. FIG. 2. Vitellaria. H3-tyrosine; 5 min pulse, 60 min chase. The imnature cells (i) are free of grains, whereas maturing (m) and some fully mature (fm) cells have many grains associated with their shell-protein globules and clusters. Some fully mature cells (fml) do not have grains. x5000. Unstained phase contrast. FIG. 3. Vitellaria. Ethylene glycol dehydration H”-phenylalanine; 5 min pulse, 0 min chase. A silver grain (arrow) is associated with the nurse cell cytoplasm, but not with the granular endoplasmic reticulum (ER) or shell globules (S ) of the adjacent vitelline cell. X50,000. FIGS. 4, 5. Vitellaria. Ethylene glycol dehydration. H3-tyrosine; 5 min pulse, 10 min chase. Grains (arrows) lie over the granular endoplasmic reticulum (ER, Fig. 4) and the Golgi region (GC, Fig. 5) and its condensing vacuoles (V). Grains are absent over shell globules (S). Fig. 4, x37,500; Fig. 5, X50,000.
22
R. E. B. HANNA
AUTORADIOGRAPHY
IN
F.
hf?patiCa
drated in alcohol, and embedded in Araldite for electron microscopy. No light microscope autoradiograms were prepared. The important features of the experimental design are summarized by Table I. c. Preparation of light microscope autoradiograms. lprn sections of the slices embedded in GMA were cut with glass knives using a Reichert Om U2 ultramicrotome. Several sections from each block were mounted, in a drop of water, near the end of an appropriately labeled microscope slide, and when all the slides were dry, they were dipped, section ends downward in a solution of 10 g of Word L4 nuclear research emulsion in 20 ml of distilled water at 45 C. Excess emulsion was allowed to drain from the slides by standing them on absorbent paper for 10 min. Then the preparations were hung in a warm dustfree atmosphere for an hour to dry, after which they were packed in air- and lighttight boxes, with silica gel present as dessicant, and were stored at 4 C for a 5-6 wk exposure period. Coating, drying, and packing of the slides was carried out under darkroom conditions using a Kodak minimum red safelight, mounted 3 ft from the working area. After exposure, the autoradiograms were developed using Kodak D-19 developer (5 min at 20 C ) , washed with 1% acetic acid, and fixed using Ilford “Hypam” fixer (1 in 10 dilution for 5 min). The slides were examined unstained, or lightly stained with 1% toluidine blue (30 set at room temperature), using a Leitz orthomat microscope and camera attachment with phase contrast illumination. Micrographs were taken at a magnification of x 900 using Ilford Pan F black and
FIGS. 6, 7, 8, 9. Vitellaria. Ethylene glycol 5 min pulse, 20 min chase; Fig. 8: H”-leucine; occur over granular endoplasmic reticulum (El%, associated with condensing vacuoles (V) of the or over shell globules (S, Fig 9). Figs. 6, 7, and
VITELLINJZ
CELLS
23
white film, or Agfachrome 50L professional color film. d. Preparation of electron-microscope autoradiograms. The method used in this investigation was based on the “loop” technique of Caro and Van Tubergen ( 1962), which is one of the simplest techniques available and has been used successfully in a number of important studies (Caro and Palade 1964, Shannon and Bogitsh 1971). Details of the technique are given in Hanna (1975) and, therefore, will not be given here. RESULTS
a. Incorporation of amino acids. Light microscope autoradiograms of slices pulselabeled with H3-tyrosine showed that this precursor had been readily incorporated by developing vitelline cells. The silver grains were concentrated around and over the shell-protein globules, which were normally at the periphery of the cells. H3-phenylalanine and H3-leucine had also been incorporated by vitelline cells, but labeling was not as heavy as it was with H3tyrosine. No activity was detected in the vitelline cells of slices labeled with H3methionine. In material from short chase periods (O-20 min), the immature vitelline cells and those in which shell-protein globules had just begun to appear had many silver grains over their cytoplasm. More mature cells also had grains around their shell globules, but fully mature cells with large glycogen stores had no grains (Fig. 1). After 45 and 60 min chase periods, however, the immature cells and those in the early stages of shell-protein synthesis had no or very few grains, whereas maturing cells had very many grains around and
Figs. 6, dehydration. 5 min pulse, 20 min Figs. 6 and 9), nurse Golgi complex (Figs. 8, x50,000; Fig. 9,
7, and 9: H3-tyrosine; chase. Grains (arrows) cell cytoplasm (Fig. 7), 8 and 9) and close to ~20,000.
24
R.
E.
B.
HANNA
AUTORADIOGRAPHY
IN
F.
over their shell globules; fully mature cells had few or no grains (Fig. 2). Electron microscope autoradiograms of slices pulse-labeled with HVyrosine, H3phenylalanine, and H3-leucine (but not those labeled with H3-methionine), showed labeling of the vitelline cells, approximately two to five silver grains per cell. In material removed immediately after the 5 min pulse, silver grains were present in the cytoplasm of nurse cells and the granular endoplasmic reticulum (GER) of vitelline cells, but not elsewhere in the latter (Fig. 3). After a 10 min chase, grains occurred over the nurse cell cytoplasm, and over the GER and condensing vacuoles of the Golgi complex of vitelline cells (Figs. 4 and 5) ; grains were not present over shell-protein globules. After 20 min, grains were present over all the sites listed previously (Figs. 6-g)) but also lay over shellprotein globules and clusters (Fig. 10). After 30 and 45 min chase periods, many grains occurred over shell-protein globules and clusters (Figs. 11 and 12), but were rare over GER and Golgi complexes. Silver grains were never found over glycogen areas or nuclei, and control tissue was always free of autoradiographic reaction. b. Incorporation of monosaccharides. ,Only those vitelline cells in which glycogenesis was occurring were labeled by H3galactose and H3-glucose. The immature cells, which were still synthesizing protein
hepaticu
VITELLINE
CELLS
25
globules, had no activity associated with them (Figs. 13 and 15). Furthermore, the nuclei, GER, and protein clusters of labeled cells were generally free of autoradiographic reaction, all the activity being present in the glycogen storage areas and yolk bodies (Figs. 13-16). The maturing cells, even those within a single follicle, showed great variation in the amount of “H-galactose incorporation (Fig. 13). The most mature cells, which were packed with glycogen, had few silver grains associated with them and in follicles where many of the cells had reached this stage, the regions between the vitelline cells (nurse cell protrusions) had many grains in their cytoplasm (Fig. 14). The distribution of labeling in the vitelline follicles did not vary with the length of time for which the slices had been exposed to the monosaccharide precursors, but the density of Iabeling over the glycogen regions was greater in material which had received longer incubations. As in the amino acid experiments, control material, which had not been exposed to radioactive precursors, showed no autoradiographic reaction in the vitelline cells. DISCUSSION
a. Shell-protein synthesis. Of the three tritiated amino acids incorporated into shell protein by immature vitelline cells,
FIG. 10. Vitellaria. Ethylene glycol dehydration, H3-tyrosine; 5 min pulse, 20 min chase. Grains (arrows) are next to or over shell globules (S ). X50,000. Fm. 11. Vitellaria. Ethylene glycol dehydration. H3-phenylalanine; 5 min pulse, 30 mm chase. Two grains (arrows) are over the shelLglobule cluster (S ). ~50,000. FIG. 12. Vitellaria. Ethylene glycol dehydration. H3-tyrosine; 5 min pulse, 45 min chase. Many silver grams (arrows) are over the shell-globule cluster (S ). X50,000. FIG. 13. Vitellaria. Ha-galactose, 5 min accumulation. The vitelline cells have incorporated different amounts of label during the short incubation. Maturing cells (m), which are carrying out glycogenesis, are heavily labeled, whereas immature cells (i) have no activity and mature cells (ma), in which glycogen synthesis is almost complete are lightly labeled. s, unlabeled shell protein. Toluidine blue, phase contrast. X3300. FIG. 14. Vitellaria. H3-galactose, 60 min accumulation. Most vitelline cells in this follicle were nearly mature (ma) when incubated and show little activity. Maturing cells (m ), however, are heavily labeled and silver grains also occur in the nurse cell cytoplasm (arrows) between the nature cells. Toluidine blue, phase contrast. X28,000.
26
R. E. B. HANNA
AUTORADIOGRAPHY
IN
F. hepaticu
H3-tyrosine was the most readily used. This finding agrees with the reports of Thorsell, Bjorkman, and Appelgren (1966) on F. hepatica and Nollen (1968) on Philophthalmus megabrus, and is not surprising in view of the fact that di-tyrosine links are partly responsible for the cross linking, and hence the resistance, of F. heputica egg shells ( Ramalingam 1973). In electron micrograph autoradiograms, silver grains appeared in the following order with time: nurse cell cytoplasm, GER, and condensing vacuoles of the Golgi complex of vitelline cells, and finally shell protein globules and clusters. With long chase periods, especially the 45 min sample, the number of grains over shell protein globules and clusters was much greater than at earlier times. This sequence in the appearance of grains with time strongly suggests that the route of shell protein synthesis and formation of shell globules is similar to that which occurs in gut cell of this fluke (Hanna 1975)) and in vertebrate tissues (Caro and Palade 1964). This study, therefore, lends support to the suggestions of Irwin (1970), and Irwin and Threadgold ( 1970) that shell protein is synthesized by the GER; is transferred to the Golgi complex for condensation; and migrates, in membrane-bound packages, to the cell periphery. Clusters of shell globules accumulate in this region, each cluster being held intact by an enveloping membrane. The activity detected in the nurse cells was probably due to incorporation of labeled molecules into structural proteins or cytoplasmic enzymes. If precursors pass through these cells prior to entering the vitelline cells they probably do so in a
FIG. 15. Vitellaria. storage areas ( G and immature cells (i) are them and the parenchyma x8000. FIG. 16. Vitellaria. glycogen stores (G and plasmic reticulum (ER)
VITELLINJI
27
CELLS
soluble form, and so would not be detectable by the present techniques. Light microscope autoradiography showed that many of the labeled cells from long chase periods were more mature than those from short chase periods. This indicates that the young cells were the most active in protein synthesis, and that during the 60 min chase period these cells became considerably more mature, and produced large amounts of shell protein. b. Glycogen synthesis. H3-galactose and H3-glucose were both incorporated readily into those vitelline cells which were producing glycogen. The variations noted in the density of labeling, between different cells in the same follicle, were probably due to variations in the rate of glycogen synthesis. The precursors were most readily incorporated by those cells in the early stages of glycogen production, while mature cells, which were already packed with glycogen stores, incorporated little, if any. The H3-galactose labeling in nurse cell regions of follicles containing many mature vitelline cells may have been due to the deposition of glycogen, using precursors which had been intended for the vitelline cells but were no longer needed by these due to the cessation of glycogenesis. No matter which incubation periods were used, H3-glucose labeling occurred throughout the glycogen regions of the cells incorporating these precursors, and some activity was also present in degenerating GER in the yolk bodies. This supports the view that glycogen deposition occurs mainly in the proximity of preformed glycogen, which might act as
Hs-galactose, 45 min accumulation. Silver grams lie over glycogen arrow) in mature vitelline cells with shell-globule clusters (S ). The unlabeled but label (arrow) appears in the nurse cell (N) between (P). m is a maturing cell which has not yet started glycogenesis. 10 min chase. The label Ha-glucose, 20 min pulse, arrows) in the vitelline cell. No activity is present in or shell globules ( S ). ~20,800.
is confined to granular endo-
28
R. E.
B. HANNA
a “template” for further synthesis (Coimbra and Leblond ( 1966) for rat liver). Although the onset of glycogen synthesis in the vitelline cells is preceded by degeneration of the GER and is accompanied by growth (both of which allow space for glycogen storage), no evidence was found for the dirct participation of any cell organelle in this process. It therefore appears that glycogenesis is mediated by cytoplasmic enzymes in these cells. The ability of F. hepatica to use galactose in glycogen synthesis implies that the glycogenic enzymes are not specific for glucose or that galactose can be converted to glucose in this animal. It is the author’s intention to discuss this phenomenon in detail in a subsequent paper. REFERENCES 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. CARO, L. G., AND VAN TUBERGEN, R. P. 1962. High resolution autoradiography. I. Methods. Journal of Cell Biology 15, 173-188. COIMBRA, A., AND LEBLOND, C. P. 1966. Sites of glycogen synthesis in rat liver cells as shown by electron microscope radioautography after administration of glucose-Hs. Journal of Cell Biology 30, 151-175. HANNA, R. E. B. 1975. Fasciola hepatica: An
electron microscope autoradiographic study of protein synthesis and secretion in gut cells in tissue slices. Experimental Parasitology, in press. HANNA, R. E. B., AND THREADGOLD, L. T. 1975. Development of an in vitro technique for cytological investigations of slices of Fasciola hepatica: Evaluation by morphological criteria. International Journal for Parasitology, in press. IRWIN, S. W. B. 1979. Some aspects of egg production in Fasciola hepatica. Ph.D. Thesis, The Queen’s University, Belfast. IRWIN, S. W. B., AND THREADGOLD, L. T. 1970. Electron microscope studies on Fasciola hepatica. VIII. The development of the vitelline cells. Experimental Parasitology 28, 399411. NOLLEN, P. M. 1968. Uptake and incorporation of glucose, tyrosine, leucine, and thymidine by adult Philophthalmus megabrus (Lost 1914) (Trematoda), as determined by autoradiography. Journal of Pamsitology 54, 295304. RAMALINGAM, K. 1973. The chemical nature of the egg-shell of the liver fluke, Fasciola
hepatica. ology SHANNON,
International
3, 67-75. W. A.,
Megalodiscw
Journal
for Pam&-
AND
BOGITSH, B. J. 1971. Comparative radioof glucose Ha and galactose Hx
temperatus.
autography incorporation. Experimental Parasitology 29, 369319. THORSELL, W., BJORKMAN, N., AND APPELGREN, L. E. 1966. Radioautographic studies on the ovary and vitelline glands of Fasciola hepatica after short in vitro incubation with some amino acids. Zeitschrift fur Parasitenkunde 28, 108-115.