Morphological studies using both light and electron microscope were carried out with the aim of characterizing cells present in the larval and adult pancreas of Xenopus laevis. The following cell types have been seen: (1) exocrine cells, with a very well developed r.e.r. (rough endoplasmic reticulum), well defined Golgi complexes and numerous large secretory granules (A cells) ; (2) cells without either r.e.r. or secretory granules but with a large number of well developed mitochondria (B cells); (3) endocrine cells often clustered in the typical islets and with small membrane-coated granules showing a very dense central core surrounded by a light halo (C cells).

During development, the aspect is seen to change from an unorganized tissue in which the acinar structures are still not clearly visible (stage 42), to a more organized form in which the exocrine cells (A cells) are seen to be arranged around the lumen of the acinus together with some B cells.

At the stages 54–56, an increasing number of acini surrounded both by A and B cells was observed. At about stage 61, large quantities of necrotic cells were seen and it became more difficult to individualize the acinar organization found in the preceding stages. Finally, there are no necrotic cells in the adult but only A, B cells which are organized in well developed acinar structures and C cells.

The investigation also included a study of some pancreatic enzymes (lipase and amylase) synthesized during larval life. Lipase activity shows a peak at stage 54–56 in which the most well organized tissue of the entire larval life was observed. The activity then decreases, reaching a minimum at stage 66, after which it rapidly rises. Maximum amylase activity occurs at stage 51 after which there is a decrease, to a minimum at stage 66. The activity then remains at constant level.

Correlations between morphological and functional data, at various stages of the secretory process of pancreatic exocrine cells, have already been demonstrated (Warshawsky, Le Blond & Droz, 1963; Jamieson & Palade, 1967 a, b, 1971).

Numerous investigations have been performed on the development of the pancreas with relation to its histological differentiation and its functional capacity during morphogenesis, and morphological observations, under in vivo and in vitro conditions, have been correlated with biochemical results (Munger, 1958; Schweisthal, Geas & Wells, 1963; Kallman & Grobstein, 1964; Dieterlen-Lièvre, 1965, 1970; Przybylski, 1967; Wessels & Evans, 1968; Parsa, Marsh & Fitzgerald, 1969; Pictet, Clark, Williams & Rutter, 1972; Track et al. 1972). Enzymic rates in the different stages of development have been correlated with the aspects of the rough endoplasmic reticulum (r.e.r.) and with the size and number of the secretory granules. Few data exist on the morphological aspect of the pancreas in vivo (Beaumont, 1968, communication) and in vitro (Pouyet & Beaumont, 1975) and on the synthesis of secretory proteins and intracellular transport in Amphibians, during larval and adult life (Urbani, 1957, 1962; Gillois & Beaumont, 1964; Slot & Geuse, 1974).

In the present investigation an ultrastructural and functional analysis was performed on modifications in the Xenopus laevis pancreas, especially at metamorphosis during which a different pattern of feeding, i.e. from a herbivorous to a carnivorous diet, takes place. Observations have been integrated with biochemical data obtained from amylase and lipase determination in the pancreas.

Several adult Xenopus laevis were mated by injecting males for 3 days, with 300 i.u./die of pregnyl, diluted with tap water. On the third day, females received a single injection of 600 i.u. Throughout treatment, the animals were kept in a bath of well water, at a temperature of about 20 °C.

Fertilized eggs were placed in a bath of about 6001. at a constant temperature of 21 °C. During larval life, the animals were fed Nettle powder.

Light microscopy

Pancreas were removed and fixed in Susa for 4 h and in Dubosq-Brasil for 24 h at room temperature. Tadpoles were cut into 6 μm sections, which were stained with haematoxylin and eosin for histological studies.

Electron microscopy

Pancreas were removed and fixed according to one of the following methods:

  1. Fixation in 1 % OsO4 in 0·013 phosphate buffer, pH = 7·4 (Millonig, 1962) for 1 h, at 4 °C.

  2. Prefixation in 3 % glutaraldehyde in 0·013 M phosphate buffer, pH = 7·4 for 45 min, at 4 °C; rinsing in the same buffer for 30 min, at 4 °C ; postfixation in 1 % OsO4 in 0·013 M phosphate buffer, pH = 7·4 for 1 h, at 4 °C.

The specimens were dehydrated in ethanol and embedded in Epon-Araldite. Thin sections, cut with an LKB Ultratome III microtome, were stained with uranyl acetate and lead citrate (Venable & Coggeshall, 1965) and examined under a Siemens Elmiskop IA electron microscope.

Biochemical determination

Pancreas were weighed and 1 % (w/v) homogenate was prepared in distilled water, using a glass Teflon homogenizer. All operations were performed at 4 °C. The homogenate was centrifuged 30 min at 15000 g.

Lipase activity was determined (by the method of Desnuelle, Constantin & Baldy, 1955), employing 5·0 ml of triolein emulsion in gum arabic with 1 mM-NaCl, 5 mM-CaCl2 and Na taurocholate (as activator, according to Broekerhoff, 1971) in a final volume of 15 ml at pH = 8· 0 at 25 °C, by continuous titration with a Radiometer TTA 2B and Titration complex TTA3. The homogenate was added to the substrate in a volume not exceeding 200 μ and the fatty acids produced were neutralized with 0·01 M-NaOH. Enzyme activity is expressed as u. per mg protein. One unit equals 1 μmol of fatty acids produced per min.

Amylase activity was determined (according to the method of Smith & Roe, 1949) employing 3–4 % starch as substrate. Enzyme activity is expressed as u. per mg protein. One unit equals 1 mg substrate changed per min.

Proteins were determined (by the procedure of Lowry, Rosebrough, Farr & Randall, 1951) using human serum-albumin as standard.

At stage 35–36 the pancreas anlage can be seen as a thickening of the dorsal midgut wall. At about stage 39–40 the two ventral pancreas anlagen, joining that of the dorsal anlage, can be distinguished. Only at stage 40 is the final localization in front of the stomach reached (Niewkoop & Faber, 1956).

Stage 42

Under the light microscope, the pancreas is seen to be very close to the gut. The tissue does not appear to be very compact: some large cavities are found between the cells, which show a pale cytoplasm (Fig. 1 A).

Fig. 1.

Light micrographs. Haematoxilin and eosin staining. (A) Larval stage 42. The acinus is not yet organized. G = gut wall. (B) Larval stage 48. Some developed acini, surrounded by connective tissue septa, are visible. S.D. = secretion ducts. (C) Larval stage 54–56. The typical acinar arrangement is recognizable. A = acinus.

Fig. 1.

Light micrographs. Haematoxilin and eosin staining. (A) Larval stage 42. The acinus is not yet organized. G = gut wall. (B) Larval stage 48. Some developed acini, surrounded by connective tissue septa, are visible. S.D. = secretion ducts. (C) Larval stage 54–56. The typical acinar arrangement is recognizable. A = acinus.

Under the electron microscope, secretory cells, showing large granules and a well developed r.e.r., are also observed. These cells, referred to as A cells, have a regular prismatic shape and are separated from the surrounding cells by a very narrow intercellular space that in some cases is electron-dense (Fig. 2 A). In several sites, the plasma membranes of two adjacent cells separate, resulting in irregular spaces limited by junctional complexes. Ther.e.r. appears in the form of numerous roundish vesicles of different size, sometimes in continuity with one another. The Golgi complex is well developed : it occupies a large area and is surrounded by numerous vesicles of the smooth reticulum. There are a large number of secretory granules occupying a vast portion of the cytoplasm: they show a typical shape and are very electron-dense (Figs. 2C and 3 A). At this stage, ‘lamellae annulatae’ arranged in small clusters of two-three units are visible near the nuclear envelope: the terminal portions are considerably swollen and bear ribosomes (Figs. 2A and 3B).

FIGURE 2.

Electron micrographs. Larval stage 42. OsO4 fixation. Uranyl acetate and lead citrate staining. (A) Portion of some A cells. The rough reticulum is organized in roundish vesicles. Mitochondria are few and large. A cluster of’lamellae annulatae’ (L. A.) is visible closeto the nucleus, which shows heterochromatic and euchromatic masses. (B) C cells. Large Golgi complex (G.C.) with several smooth vesicles and many mitochondria localized in the cytoplasm and some small secretion granules (g), characterized by a very dense central core, surrounded by a clear halo, can be seen. (C)View of acinar structure. L = lumen; A = A cell; B = B cell. Several vesicles are visible in the apical cytoplasm of B cells (arrows).

FIGURE 2.

Electron micrographs. Larval stage 42. OsO4 fixation. Uranyl acetate and lead citrate staining. (A) Portion of some A cells. The rough reticulum is organized in roundish vesicles. Mitochondria are few and large. A cluster of’lamellae annulatae’ (L. A.) is visible closeto the nucleus, which shows heterochromatic and euchromatic masses. (B) C cells. Large Golgi complex (G.C.) with several smooth vesicles and many mitochondria localized in the cytoplasm and some small secretion granules (g), characterized by a very dense central core, surrounded by a clear halo, can be seen. (C)View of acinar structure. L = lumen; A = A cell; B = B cell. Several vesicles are visible in the apical cytoplasm of B cells (arrows).

FIGURE 3.

Electron micrographs. Larval stages 42 (Fig. A/B) and 48 (Fig. C/B) OsO4 fixation. Uranyl acetate and lead citrate staining. Portion of A cell and two B cells. The arrow indicates an irregular space between the cells. Many small vesicles are visible around the Golgi complex: they are interpreted as elements of smooth endoplasmic reticulum. High magnification of ‘lamellae annulatae’ in the cytoplasm of an A cell. A cluster of two elements showing the typical pattern is visible. Ribosomes are attached to the terminal vesicles (arrows). Golgi complex in the cytoplasm of an exocrine cell (A cell). The peripheral vesicles are filled with electron-dense material (arrows). Portion of an exocrine cell (A cell) and some endocrine cells (C cells); some typical secretion granules are visible in the C cell (arrow).

FIGURE 3.

Electron micrographs. Larval stages 42 (Fig. A/B) and 48 (Fig. C/B) OsO4 fixation. Uranyl acetate and lead citrate staining. Portion of A cell and two B cells. The arrow indicates an irregular space between the cells. Many small vesicles are visible around the Golgi complex: they are interpreted as elements of smooth endoplasmic reticulum. High magnification of ‘lamellae annulatae’ in the cytoplasm of an A cell. A cluster of two elements showing the typical pattern is visible. Ribosomes are attached to the terminal vesicles (arrows). Golgi complex in the cytoplasm of an exocrine cell (A cell). The peripheral vesicles are filled with electron-dense material (arrows). Portion of an exocrine cell (A cell) and some endocrine cells (C cells); some typical secretion granules are visible in the C cell (arrow).

Cells were also observed with very little r.e.r. and no secretory granules: these are referred to as B cells. They have a characteristic shape; most of them have irregular processes protruding between the A cells (Fig. 2C). The cytoplasma contains numerous free ribosomes often clustered in ‘rosettes’. The Golgi complex is well developed and abundant in large cisternae in some of which a slightly electron-dense material is present. There are also more large mitochondria than in other pancreatic cells.

Finally, isolated or clusters of about two or three cells may be present (Fig. 2B). The cytoplasm of these cells is very rich in free ribosomes, rough vesicles and small membrane-coated granules, which are characterized by a clear halo. These are referred to as C cells.

Stage 48

Pancreas organization appears slightly more complex than in the previous stages. A few acini can be detected, under the light microscope (Fig. 1B).

At the ultrastructural level, the most significant modification is found in the r.e.r. of the A cells. Frequent transitional forms are observed between the vesicle structures and elongated cisternae. There is also a substantial increase in free ribosomes, which makes the cytoplasm slightly electron-dense (Fig. 3D). A very electron-dense material is also visible within some vesicles of the Golgi complex. B and C cells show no changes at this stage (Fig. 3C, D).

Stage 54–56

Pancreas is very well organized: acini are differentiated and secretory structures are visible under the light microscope. Connective tissue septa are well developed (Fig. 1C).

The characteristic of both A and B cells at this stage, revealed by ultrastructural studies, is the presence of lumina which give the tissue a typical pancreatic acinar pattern. Each acinar lumen is surrounded by both A and B cells, connected to one another by typical junctional complexes (Fig. 4 A). Around these lumina, A cells with numerous microvilli show a pyramidal shape, similar to secretory cells in the adult tissue (Fig. 4A).

FIGURE 4.

Electron micrographs. Larval stages 54–56. OsO4 fixation. Uranyl acetate and lead citrate staining. (A) Exocrine A cells and B cells. The cells surrounding the lumen are connected by junctional complexes (arrows). G.C. = Golgi complex ;C = centriol. The* indicates a series of microvesicles near the plasma membrane of the B cell. (B)Endocrine islet. Secretion granules are present within the endocrine cells. (C) A and B cells. Infoldings of the plasma membrane of the B cell within the adjacent B cell are visible. Note that the invaginations seem to elongate into a series of micro vesicles (arrows).

FIGURE 4.

Electron micrographs. Larval stages 54–56. OsO4 fixation. Uranyl acetate and lead citrate staining. (A) Exocrine A cells and B cells. The cells surrounding the lumen are connected by junctional complexes (arrows). G.C. = Golgi complex ;C = centriol. The* indicates a series of microvesicles near the plasma membrane of the B cell. (B)Endocrine islet. Secretion granules are present within the endocrine cells. (C) A and B cells. Infoldings of the plasma membrane of the B cell within the adjacent B cell are visible. Note that the invaginations seem to elongate into a series of micro vesicles (arrows).

The r.e.r.is generally organized in parallel cisternae which are often connected. Other areas contain unconnected roundish vesicles. The appearance of the Golgi complex and secretory granules is the same as described at stage 48.

The B cells have elongated plasma membrane invaginations at the endings of which many microvesicles are seen parallel to the cell borders. The invaginations often form infolding branching structures which come into very close proximity with the peripheral vesicles of the Golgi complex (Fig. 4C).

C cells are easily detected since not only are they almost completely filled with secretory granules but also since they often form typical islets (Fig. 4B).

Stage 61

The specific features of the organ are: disappearance of the acinar structure which was characteristic of the previous stages and significant modifications in the cell ultrastructure. There are many necrotic cells interspersed throughout the tissue and sometimes complex digitations appear between adjacent plasma membranes (Fig. 5B). A cells are different from the B or C cells on account not only of their localization, but also on account of the marked development of the r.e.r., whichis generally made up of round vesicles or elongated cisternae, occasionally very swollen and separated by electron-dense cytoplasm. Lipid droplets are often observed, surrounded by r.e.r. which has a typical concentric pattern (Fig. 5 A).

FIGURE 5.

Electron micrographs. Larval stage 61 (Fig. A and B) and adult (Fig. C). Uranyl acetate and lead citrate staining. OsO4 fixation. A lipid droplet (L.D.) surrounded by r.e.r. is visible in a exocrine cell. The arrangement of the r.e.r. cisternae is very similar to that of adult. A and C cells. The exocrine cell (A cell) is markedly necrotic and the endocrine C cell shows a more vacuolated cytoplasm than in previous stages. A cell’s r.e.r. is organized in a concentric pattern as in larval stage 60.

FIGURE 5.

Electron micrographs. Larval stage 61 (Fig. A and B) and adult (Fig. C). Uranyl acetate and lead citrate staining. OsO4 fixation. A lipid droplet (L.D.) surrounded by r.e.r. is visible in a exocrine cell. The arrangement of the r.e.r. cisternae is very similar to that of adult. A and C cells. The exocrine cell (A cell) is markedly necrotic and the endocrine C cell shows a more vacuolated cytoplasm than in previous stages. A cell’s r.e.r. is organized in a concentric pattern as in larval stage 60.

B cells appear to have decreased in number, although their characteristics remain in the same.

C cells do not undergo any evident change either in number or in appearance, except for the presence of a more vacuolated cytoplasm than in earlier stages (Fig. 5B).

Adult pancreas

The characteristics of the pancreas at this stage are the definite acinar organization and the concentric arrangement of the cisternae in the r.e.r. within the A cells. While there are fewer B cells than in previous stages, C cells are about the same (Fig. 5C).

Biochemistry

Results of the enzymic determinations are shown in Fig. 6. A continuing increase in lipase activity is observed during early larval development, with a peak of maximal activity at about stage 55.

Fig. 6.

Specific activities of pancreatic lipase ( × ) and amylase ◼at different larval stages and in adult life of Xenopus laevis. The metamorphosis takes place between stages 59–60 and stage 66. Each point is the mean of triplicate experiments, performed on the same homogenate, ± S.E.M. About 30 animals in the early stages of development and about 10 animals in the latest ones, were used for each determination. For experimental details, see text.

Fig. 6.

Specific activities of pancreatic lipase ( × ) and amylase ◼at different larval stages and in adult life of Xenopus laevis. The metamorphosis takes place between stages 59–60 and stage 66. Each point is the mean of triplicate experiments, performed on the same homogenate, ± S.E.M. About 30 animals in the early stages of development and about 10 animals in the latest ones, were used for each determination. For experimental details, see text.

From this stage until advanced metamorphosis, activity rapidly decreases and almost disappears at about stage 66. At the end of metamorphosis, activity gradually begins to increase reaching a level equal to that in adult life, which is in keeping with the results obtained in the developing pancreas of the chick (Kulka & Duskin, 1964).

Amylase activity does not show the same behaviour as lipase. Starting from the early stages, the activity decreases until stage 53, showing an insignificant peak at stage 55 and falls to its lowest level at about stage 66. This low activity remains fairly uniform in adult life which, also in this aspect, is unlike lipase.

Analysis of the results obtained appears to suggest that morphological and biochemical data are correlated. In fact the activity of the two enzymes reaches a peak at stages 51 and 55, which corresponds to the highest organization level of acinar tissue (Fig. 6).

From stage 42, the secretory cells undergo a process of gradual differentiation culminating at stage 55 when the larval pancreas is at its highest level of organization, both from a histological and cytological point of view.

At this stage, A cells are arranged in the typical acinar form : the number of secretory granules and the appearance of the r.e.r. also show that these cells have reached complete structural differentiation (Fig. 4 A). However, before the regular acinar structure is completely established, the cells have a basophilic cytoplasm, with free ribosomes, cisternae of the r.e.r. and well developed Golgi complexes (Figs. 2C and 3 A). From our observations it appears that pancreas in its early stages of development is already able to function : the progressive increase in lipase and amylase activity observed during development could be due both to an increase in the number of secretory cells and to an increase of cell structures responsible for synthesis and secretion.

The presence of ‘lamellae annulatae’ with bound ribosomes supports the hypothesis that the r.e.r. stems from the peripheral cisternae of the ‘lamellae’, as previously proposed by Wischnitzer (1970).

As far as concerns the B cells, two hypotheses can be advanced : (1) the fact that they are fairly numerous in the early stages of development, whereas later they tend to decrease, appears to suggest that initially they represent undifferentiated elements which are later transformed into differentiated exocrine or endocrine cells. This hypothesis would be in keeping with the ‘biphasic development model of mouse embryonic pancreas’ proposed by Pictet et al. (1972), or (2) on account of their localization in the tissue, they could be considered similar to the typical centro-acinar cells of the adult.

Their specific characteristic is that they are relatively poor in r.e.r., which means a low synthesis level. This aspect appears to lend further support to both the hypotheses proposed. In fact, the absence of a well developed r.e.r. could correspond to an early phase in a process leading to exocrine or endocrine differentiation; on the other hand it could be the final pattern of cells which would later have limited protein synthesis and secretory functions, such as the centroacinar cells.

In addition, the plasma membrane invaginations of B cells and the vesicles emitted from them, tend to suggest that they play a key role in the development of membrane structures within the cell, in view of a later differentiation. However, these vesicles could take part in the pinocytotic process (Figs. 2C and 4 A). Thus, it would appear that the B cells are involved in intra- and/or inter-cellular transport. Finally, they might also be an artifact due to OsO4 treatment, since vesicles were less frequently observed in specimens with double fixation (glutaraldehyde and OsO4) (Tormey, 1964).

One of the most significant aspects of the present study concerns the analysis of the stages prior to and immediately following metamorphosis. In fact, even if no histological and ultrastructural changes were observed in the advanced stages of development, a marked decrease in enzymic activity was demonstrated, the lowest point of which coincides with the peak of metamorphic crisis. The decrease in enzymic activity occurs when the animal takes up no food (Komáromy, Montskó, Tigyi & Lissák, 1967; Morisset & Webster, 1972). This would account for a slackening in the development process observed in the organ together with onset of necrosis of the tissue (Reeder, 1964). As far as adult life is concerned, a constant increase in lipase activity can be seen, while the amylase activity remains at very low levels (McGeachin & Porter Welbourne, 1971). This is in agreement with the variation in the diet, which is mainly carnivorous in adult life.

We are most grateful to Dr L. Conti and Dr A. Ciofi Luzzatto for their criticism and advice and to Mrs V. Autuori Pezzoli for skilful technical assistance during the course of the study.

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