TISSUE AND CELL, 1992 24 (1) 17-29 Q 1992 Longman Group UK Ltd.

CARLA FENOGLIO, GRAZIELLA BERNOCCHI and SERGIO BARNI

FROG HEPATOCYTE MODIFICATIONS INDUCED BY SEASONAL VARIATIONS: A MORPHOLOGICAL AND CYTOCHEMICAL STUDY Keywords: Frog, hibernation,

hepatocyte, cytochemistry,

glycogen distribution

ABSTRACT. A correlated morphological and cytochemical approach was employed to study frog hepatocytes in different periods of their annual cycle, including the natural hibernating period. There were considerable changes in the distribution and organization of hepatic glycogen in different phases of the annual cycle, and distribution of organelles as well. The most striking findings were glycogen storage during the prehlbernation and hibernation phases, followed by drastic glycogen depletion. Cytochemical staining of a number of enzymes (succinate dehydrogenase, lactate dehydrogenase, glucose-6-phosphate dehydrogenase, paranitrophenyl phosphatase, acid phosphatase, and glucose-6-phosphatase) involved in a variety of metabolic pathways, showed various cytoplasmic localizations and differences in intensity of the reaction products as a function of seasonality. Morphological and cytochemical data were interpreted as evidencing different functional requirements during seasonal changes in the frog.

Introduction

Hibernation provides model conditions to study frog liver adaptations that seem to allow the frog to survive the unfavourable circumstances of low temperature and prolonged starvation. Studies that have been published include that of Villani and Niso (1979) on morphological changes in hibernation artificiallyinduced by cold, and that of Brachet et al. (1971) on biochemical changes during natural hibernation. In this research, we investigate the liver of frog during different periods of the year, including natural hibernation. We compared the ultrastructural morphology of hepatocytes, their glycogen and lipid contents and the activities of enzymes linked to several metabolic pathways, to obtain information about the changes in the functional state of the hepatocyte by correlating the morphological aspect of the tissue with the cytochemical findings.

The normal ultrastructural morphology of the amphibian hepatocyte described by several investigators indicates that the abundance of glycogen deposits is a special structural aspect of this cell (Dawson, 1931; Godula, 1970; Spornitz, 1975). Liver glycogen levels in these animals can reach concentrations five to ten times those typically found in mammals (Farrar, 1972; Smith, 1950). Moreover, several natural or artificial stimuli, including metamorphosis, vitellovariations genesis, temperature and nutritional status, also affect the morphological appearance of amphibian hepatocytes (Mizell, 1965; Nicholls et al., 1968; Spiegel and Spiegel, 1970; Bennet and Gleen, 1970; Brachet et al., 1971; Villani and Niso, 1979; Duveau and Piery, 1973; Bait et al., 1979; Villani, 1980). Dipartimento di Biologia Animale e Centro per 1’Istochimica de1 C.N.R.-Piazza Botta, Pavia, Italy. Address for correspondence: Dr. Carla Dipartimento di Biologia Animale, Piazza Botta Pavia, Italy.

di Studio 10-27100

Materials and Methods

Fenoglio, lo,27100

Rana esculenta L. of both sexes were caught in their natural environment over the period of a year.

Received 15 April 1991. Revised 16 September 1991. 17

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FENOGLIO

Animals hibernating underground were caught in January (mean environmental temperature = 0” C); active frogs living in an aquatic habitat were collected in April (mean environmental temperature = 18” C), June (mean environmental temperature = 22” C), October (mean environmental temperature = 15” C). Six animals for each period were used, immediately after capture. Each group was examined by the following procedures: (1) ultrastructural observation by conventional electron microscopy, (2) morphological and histochemical light microscopy with hematoxylin-eosin, toluidine blue and PAS staining to visualize glycogen, (3) demonstration of lipid content with fluorochrome Nile red, (4) histochemical detection of some dehydrogenase and phosphatase activities. Electron microscopy

A portion of the liver from each animal was minced into small fragments (OS-l.0 mm) and fixed by immersion for 3 hr in ice-cold 1.5% glutaraldehyde in 0.05 M cacodylate buffer, pH 7.4, containing 7% sucrose, followed by post-fixation in 1% 0~0~ in the same buffer. Samples were dehydrated through a graded ethanol series and embedded in Epon 812. Ultrathin sections (ca. 600 8, thick) were contrasted with uranyl acetate and Reynold’s lead citrate and examined in a Philips 300 electron microscope operated at 60 KV. General morphology

and histochemistry

For morphological observations, sections (1 ,um thick) of epon-embedded liver samples were stained with 1% toluidine blue in borax and cryostat slices (14 pm thick) from frozen

ETAL.

tissue cubes were stained with hematoxylineosin. Cryostat sections were also used for the PAS reaction to demonstrate the glycogen content, with and without prior digestion with 0.2% a+amylase. Lipid storage was demonstrated after fluorochromization of cryostat sections with Nile red, according to the method of Fowler and Greenspan (1985). Fluorochromized specimens were observed with an epi-fluorescence Zeiss Axioplan microscope (A ext. = 480 nm) . Enzyme histochemistry

Frozen sections of liver were used for the following enzyme histochemistry: succinate dehydrogenase lactate (SDH), dehydrogenase (LDH), glucose-6-phosphate dehydrogenase (G6PDH), by the method of Lojda et al. (1979); para-nitrophenyl phosphatase (pNPPase), by the method of Mayahara et al. (1980); acid phosphatase (AcPase) by the method of Burstone (1962); glucose-6-phosphatase (GdPase), by the method of Wachstein and Meisel (1956). To avoid diffusion of the enzyme, the media to demonstrate LDH and G6PDH contained 17% (w/v) polyvinyl alcohol (PVA). For each enzyme, control sections were incubated in substrate-free media. Results Hibernating frogs collected in January Morphology

The liver parenchyma consisted of large hepatocytes arranged in laminae two cells thick, separating adjacent sinusoids. All the

Figs l-10. Hibernating frog Fig. 1. Semithin section. Large hepatocytes are filled with glycogen; cytoplasmic organelles are segregated in a restricted peribiliar area. x300. Fig. 2. Photomicrograph showing the polarity of intracellular components in four adjacent hepatocytes bordering a bile canaliculum. x6800. Fig. 3. PAS reaction. Strong reactivity due to stored glycogen is uniformly distributed in the liver parenchyma. x300. Fig. 4. Nile red fluorochromization. fluorescent lipid drops.x300.

The hepatocytes show few and peripherally distrihuted

FROG HEPATOCYTE

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MODIFICATIONS

hepatocytes showed a polarity of intracellular components (Fig. 1). The cytoplasm of all hepatocytes was filled for the most part with glycogen, so that the few organelles present in the cells (mitochondria, rough endoplasmic reticulum and lysosomes) were segregated in a restricted peribiliar area or encircling the cell nucleus. The mitochondria showed a dense matrix and few cristae. The glycogen fields showed low density and /?particles of glycogen generally predominated over a-rosette forms (Fig. 2). Occasional lipid droplets were seen.

deposits, due to G6PDH, were unevenly distributed throughout the cytoplasm of hepatocytes (Fig. 6). Weak reaction deposits were observed near the bile poles of hepatocytes stained for SDH (Fig. 7). AcPase activity, seen as fine granules, was mainly observed near the bile poles of most hepatocytes (Fig. 8). There were heavy reaction deposits in the same location in the liver section stained for G6Pase (Fig. 9). In the same period, there was a weak pNPPase activity at the cell membrane (Fig. 10). Active frogs collected iu April and June

Cytochemistry

There was intense and homogeneous PASpositivity in almost all hepatocytes of hibernating frog samples (Fig. 3). A few fluorescent spots were seen at the peripheries of cells in specimens stained with Nile red to demonstrate the lipid content (Fig. 4). Sections incubated for oxidoreductase activities showed different intensities and distributions of formazan deposits for the different enzymes. Moderate LDH activity was mainly localized in those areas of hepatocytes presumably occupied by organelles and cytoplasmic matrix (Fig. 5), i.e., near the bile pole or around the nuclei. Lighter formazan

Morphology The general organization of liver tissue was similar to that previously described, but with major morphological differences between hibernating and active frog hepatocytes. Hepatocellular volume was reduced, presumably an effect linked to the dramatic depletion of the glycogen content. The polar localization of organelles was no longer evident in these periods (Fig. 11). Variable numbers of mitochondria were scattered throughout the cytoplasm, in greater numbers and larger than those of hibernating specimens, although no measurements were made. The cistemae of

Fig. 5. LDH activity. Cells showing intense formazan deposits localized in peribiliar areas. x300. Fig. 6. G6PDH activiy. The hepatocytes show uneven and slight reactivity. x300. Fig. 7. SDH activity. Weak formazan precipitates areas. x300.

are localized in restricted cytoplasmic

Fig. 8. AcPase activity. Fine granules of reaction product are mainly localized at the bile pole of hepatocytes. x300. Fig. 9. G6Pase activity. Heavy deposits in a peribiliar location are present in the hepatocytes. x300. Fig. 10. pNPPase activity. Faint reactivity can be seen around the hepatocytes.

X300

Figs 11-20. Active frog collected in June Fig. 11. Semithin section. Small hepatocytes are almost devoid of glycogen deposits. x300. Fig. 12. Photomicrograph

of hepatocytes showing abundant cytoplasmic organelles. x6800.

Fig. 13. PAS reaction. A drastic reduction of PAS-positive areas is noticed in this section, in comparison to the hibernating frog. x300. Fig. 14. Nile red fluorochromization. drops of different sizes. x 300.

The lipid storage is represented by numerous fluorescent

I

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the rough endoplasmic reticulum were in parallel arrays, sometimes in close association with the mitochondria (Fig. 12); in some cells the rough endoplasmic reticulum was slightly dilated. Cytochemistry

There was less prominent PAS staining in liver samples from frogs caught in April and June than in those from pre- and hibernating frog specimens. However there was moderately more staining in June samples (Fig. 13) than in April samples. After the Nile red reaction, numerous scattered fluorescent drops could be seen in hepatocytes from June frogs. These were located at the periphery of the cell (Fig. 14). As for the oxidoreductase activities, in both periods there was faint .LDH reactivity distributed homogeneously in the cytoplasm of the hepatocytes, as both fine granules and diffuse reaction product (Fig. 15). G6PDH gave homogeneous and intense cytoplasmic staining, especially in June frogs (Fig. 16). Compared to the hibernating frog, there was a general increase in SDH activity in these periods, especially for June versus April (Fig. 17).

ET AL.

In the same periods there was a weak AcPase reaction in a granular pattern, in most hepatocytes (Fig. 18). There was moderate staining, distributed almost homogeneously over the parenchyma, after the G6Pase reaction (Fig. 19). There was a stronger pNPPase activity on the plasma membrane of most hepatocytes, especially at the sinusoidal surface, than in the hibernating samples (Fig. 20). Active frogs collected in October Morphology

The hepatocytes in this period exhibited a specific morphological aspect: hepatocellular volume was increased because of the large and very dense glycogen fields, (Fig. 21) in which the d-rosettes were the predominant form of glycogen organization (Fig. 22). However in spite of the abundance of glycogen, numerous organelles were still scattered throughout the cytoplasm. The mitochondria were very numerous, but small in size and with a rather dense matrix. There were numerous stacks of parallel cisternae of rough endosplasmic reticulum both throughout the cytoplasm and in proximity to the

Fig. 15. LDH activity. Weak and granular reaction product is homogeneously distributed in the hepatocytes. x300. Fig. 16. G6PDH activity. Heavy reaction product is uniformly distributed in the cytoplasm of hepatocytes. x300. Fig. 17. SDH activity. Most hepatocytes show an intense and homogeneous reactivity. x300. Fig. 18. AcPase activity. Hepatocytes

showing finely scattered granular positivity. x300.

Fig. 19. G6Pase activity. Moderate activity is distributed throughout hepatocytes. x300.

the cytoplasm of the

Fig. 20. pNPPase activity. Intense reactivity can mainly be observed in the sinusoidal regions of hepatocytes. x300. Figs 21-30. Active frog collected in October. Fig. 21. Semithin section. Large hepatocytes showing abundance of glycogen and numerous organelles scattered throughout the cytoplasm. x300. Fig. 22. Photomicrograph of hepatocytes showing abundant deposits of glycogen rosettes; numerous mitochondria and stacks of endoplasmic reticulum are dispersed in the cytoplasm. X6800. Fig. 23. PAS reaction. Very strong reactivity due to stored glycogen is evenly distributed in the liver parenchyma. x 300. Fig. 24. Nile red Buorochromization. hepatocytes. x300.

Scanty fluorescent lipid drops can be noticed in most

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FENOGLIO

nucleus (Fig. 22). A few lipid droplets were observed at the periphery of most cells. Cytochemistry

All the hepatocytes of these samples were very strongly PAS-positive (Fig. 23), with moderate Nile red labeling detected in most hepatocytes (Fig. 24). There was generally a moderate staining in hepatocytes after all three oxidoreductase reactions. In most cells formazan deposits were not homogeneously distributed throughout the cytoplasm of the hepatocytes (Figs 25, 26, 27). After the incubation for AcPase, the staining pattern within the hepatocytes resembled that of the hibernating samples, i.e., moderate amount of reaction product was mainly observed in the peribiliary zone, though several cells showed a diffuse pattern of staining (Fig. 28). There was moderate and diffuse staining in most cells after the G6Pase reaction, though some cells showed weak activity (Fig. 29). A moderate pNPPase activity was observed on the cell membrane of most hepatocytes (Fig. 30). Controls The PAS-positivity was abolished in all specimens by digestion with d-amylase. There were no reactions in samples incubated in substrate-free media. Discussion The morphological and cytochemical results of this study show marked differences in the hepatocytes of Rana esculenta in the different phases of the seasonal cycle (hibernation, activity and prehibernation).

The most striking differences are in the distribution, appearance and amount of glycogen. As we mentioned in the Introduction, there is an abundance of glycogen in the hepatocytes of several amphibians, but there is no agreement about whether or not this accumulation is subject to cyclic daily mobilization (as in most mammals) or to cyclic seasonal mobilization. Our data show clearly that stored glycogen is a larger source of energy than stored lipids for Rana esculenta, since the large amount of the material that can be detected by the PAS reaction in the prehibernation and hibernation phases is greatly depleted after hibernation. The digestion of this accumulated carbohydrate is very slow, since from October to January the areas occupied by glycogen do not decrease, although their configuration and density change. The organization of glycogen in the two typical a-rosette and /I-particles forms varies in the different periods. There are more (Yrosettes in the prehibernation phase and more o-particles in hibernation and the month immediately after it. Bait et al. (1979) found a change of glycogen from the a to the p configurations in the livers of Rana pipiens fasted in the laboratory and Segner and Braunbeck (1990) found it in the liver of the teleost Leuciscus idus melanotus at the end of winter, a period during which the animal does not feed. This might indicate, according to Bait et al. (1979), that the two configurations have different stability because of different molecular organization. However, we cannot ignore Spornitz’s (1975) contrasting results for the liver of Xenopus laevis, in which the glycogen content did not change even after 5 months of forced

Fig. 25. LDH activity. The hepatocytes are seen in several hepatocytes. x300. Fig. 26. G6PDH

activity.

Fig. 27. SDH activity.

Moderate

Faint staining

exhibit

reactivity

weak reactivity;

Fig. 30. pNPPase tocytes. X300.

activity. activity.

occurs in most hepatocytes.

Cells showing Moderate

clumpy formazan

can be seen in most hepatocytes.

Fig. 28. AcPase activity. The cells show a moderately restricted to the bile pole. x300. Fig. 29. G6Pase

ET AL.

a heterogeneous

reaction

product

positive

intensity outlines

deposits

x300.

x300. reaction

product

of staining.

mainly

x300.

the cell membranes

of hepa-

28

fasting. Spornitz, himself, suggested (1975) that the reason for this difference from the frog is that Xenopus does not naturally hibernate. Our morphological data agree well with the cytochemical data for the enzymes involved in carbohydrate metabolism, G6Pase, G6PDH and LDH. During hibernation, when glycogen almost completely fills the cell, there is also strong G6Pase activity and weak G6PDH activity. During this phase the hepatic glycogen is the metabolic energy source for the entire body and the strong G6Pase activity, which releases glucose into the circulation, would be in accord with this. In the same period, the activities of SDH and pNPPase are very weak, indicating decreased energy metabolism and decreased membrane transport. The moderate activity of LDH, an indicator of anaerobic metabolism, might be related to the lesser oxygenation in the hibernating animal. We might hypothesize that there is also a relationship between LDH activity and temperature. Crawford et al. (1990) have recently reported that the livers of the teleost Fundulus heteroclitus living in the North (Canada) have more LDH activity than the livers of the fish living in the South (Florida). In April, after the end of hibernation, the hepatocyte of the frog becomes almost completely emptied of its carbohydrate content, the cell volume is markedly decreased, and are distributed evenly the organelles throughout the cytoplasm. In addition, the rough endoplasmic reticulum and the mitochondria appear to increase in number and size. In April, and until June, there are high levels of G6PDH, SDH and pNPPase activity. These are evident signs of return of synthetic metabolism, energy metabolism and of active membrane transport. The phase of accumulation of stored material appeared to culminate about the end of October, at which time the morphological studies show the hepatocyte to be almost full of glycogen. The organelles, distributed in the cytoplasm between the large areas of glycogen, are similar in appearance to those of the hibernating frog (condensed mitochondrial matrix, cisternae of the reticulum tightly packed), and the cytochemistry supports the idea that the cells are already metabolically quiescent, with the enzyme activities moderately positive. Fur-

FENOGLIO HAL.

thermore the weak G6Pase staining indicates that the animal can still nourish itself, since it is not yet hibernating, and save the stored carbohydrate for hibernation. The differences in the lysosomal enzyme AcP during the annual cycle are reflected in the distribution more than in the amount of activity that can be demonstrated cytochemically. The morphological results agree with cytochemical results, with the epithelial layers made up of intact cells and a normal lysosome content, whose distribution however, varies according to the different periods. Cell debris is seen only occasionally inside the sinusoids, primarily in the winter. In the same period, the other organelles do not appear to have degenerated, but to be only temporarily inactive, in agreement with the decrease in cell metabolic activity demonstrated cytochemically. We, therefore can deduce that the hepatocytes of Rana esculenta do not degenerate during hibernation. Neither Spornitz (1975) nor Bait et al. (1979) found cellular abnormalities in Xenopus laevis or Rana pipiens after several months (2-5) of fasting. Therefore, the abnormalities observed by Duveau and Piery (1973) in hepatocytes of Rana esculenta (fatfilled vacuoles, pyknotic nuclei, abnormal mitochondria) were probably due to the prolonged period of hibernation (18 months) imposed by the investigators. We found no significant differences in the aspects we studied between individual frogs nor between the sexes. The cells were morphologically and cytochemically homogeneous in January, one extreme of the cycle, and the same was true at the other extreme, in June. In the intermediate periods (April and October), they were less homogeneous. Some of the changes that we have described have also been described by other investigators for selected fish (Valtonen, 1974; Quaglia, 1976; Saez et al., 1984; Barni et al., 1985; Segner and Braunbeck, 1990) during seasonal and biological cycles. We wish to emphasize that among lower vertebrates there are many species-dependent factors (i.e. vitellogenesis, metamorphosis, feeding) that can interact with the modifications of the hepatocytes and contradictory findings should be evaluated with this in mind.

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MODIFICATIONS

Acknowledgements We are very grateful to Professor G. Gerzeli for critically reading the manuscript. We also

gratefully acknowledge the technical assistance of R. Vaccarone, G. Balza and F. Vai. This study was supported by! grants from Italian MURST (40% and 60%).

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