Acta Zoologica (Stockh.), Vol. 69, NO. 4, pp. 225-230. 1988 Printed in Great Britain

000 1-7272/88$3.OO+ .OO Pergamon Press plc

The Royal Swedish Academy of Sciences

Autoradiographic Investigations on Cell Proliferation in the Digestive Mucosa of Fishes Robert Kiss,'.' Yvan de Launoir, '.' Ceorges Lenglet' and Andre Danguy'

'Laboratoire de Biologie Animale et d'Histologie Comparte, Facultt des Sciences, Universitt Libre de Bruxelles, 50 avenue Franklin Roosevelt, B-1050 Bruxelles, Belgium, and 'Laboratoire d'Histologie, Facultt de Mkdecine, Universitt Libre de Bruxelles, 2 rue Evers, B-1000 Bruxelles, Belgium (Accepted for publication 21 October 1987)

Abstract Kiss, R., de Launoit, Y . , Lenglet, C., Danguy, A. 1988. Autoradiographic investigation on cell proliferation in the digestive mucosa of fishes. (Laboratoire de Biologie Animale et d'-Histologie Comparte, FacultC des Sciences, and Laboratoire d'Histologie, Facultt de Mtdecine, Universitt Libre de Bruxelles, Bruxelles, Belgium.)-Acra 2001. (Stockh.) 69. 225-230.

The labelling index (TLI) of the digestive mucosa of some fish species was determined following a pulse labelling with tritiated thymidine ([.'H]TdR) and light microscopic autoradiography. In the oesophageal epithelium, proliferation was observed to occur in non mucus-secreting cells. In the intestine, both undifferentiated and absorptive cells incorporated pH)TdR within 1 h after injection. Statistically significant differences in [3H]TdR incorporation were observed between the upper intestine region and both the middle and lower parts on the one hand, and between the middle and lower parts on the other hand. Mucus-secreting cells seemed unable to proliferate. In the stomach, significantly fewer labelled nuclei were counted; they were located in the isthmus epi- thelium. No significant difference was observed between the TLI of these regions in the different species.

Andrk Dunguy, Laboratoire de Biologie Aniniale er d'Hisrologie Coniparee CP 160. . FacultP des Sciences, UniversitP Libre de Bruxelles. 50 avenue Franklin Roosevclr, B-I050 Bruxelles. Belgium.

Introduction

In mammals it is well established that the epi- thelial lining of all portions of the alimentary tract undergoes continuous renewal. Cell kinetics in the gastrointestinal mucosa has been studied chiefly in a variety of murine strains (Hugues et al. 1958, Leblond and Messier 1958, Lipkin and Quastler 1962, Cameron and Greulich 1963, Pil- grim 1964, Cairnie et al. 1965, Dormer and Moller 1968, Schultze et al. 1972, Kovacs and Potten 1973, Al-Dewachi et al. 1975, Hattori and Fujita 1976, Cheng and Bjerknes 1982) and some other species, including man (Bertalanffy and Nagy 1961, Willems and Galand 1970, Bleiberg et al. 1971, 1985). However, little attention has been paid to cell proliferation of the gut in lower vertebrates (O'Steen and Walker 1960, Patten 1960, Wurth and Musacchia 1964, Garcia and Johnson 1972, Gas and Noaillac-Depeyre 1974, McAvoy and Dixon 1977, Kiss et al. 1986b) and for this reason we undertook this study. This paper deals with radioautographical data on the incorporation of tritiated thymidine (["H]TdR) by cells of various parts of the digestive tract epithelium of 10 fish species.

Thymidine labelled with tritium is universally used to label specifically any new DNA that is being synthesized (in phase S of the cycle) (Rogers 1979). The proliferation activity of a cell population can be assessed in a quantitative way by estimating the percentage of cells that have incorporated ['HITdR after a pulse exposure.

Materials and Methods

1. Animals

Specimens of 10 species (see Table 1) were purchased from a local tropical fish wholesaler. Prior to exper- imentation they were fed Tetra Min and frozen brine shrimp and kept in aerated aquaria filled with aged tap water at 22.C25.0"C under a 12 h light-12 h dark photoperiod. The fishes were acclimated over three weeks.

Four or five specimens of each species were used. In order to avoid the effects of an eventual 24 h cycle variation in the mitotic rates of the digestive tract (Chiakulas and Scheving 1961) the animals were sacri- ficed at the same time during the day (10.00-11.00 am).

225

226 Robert Kiss et al.

Table 1. List of the investigated species

Family Species

Standard Weight length (g) (mm)

Cyprinidae Barbus sp. (1) 19-21 Danio malabaricus (2) 14-18

Characidae Gymnocorymbus ternetzi (3) 20-22 Hemigramrnus caudovittatus (4) 25-28 Moenkhausia sanctae filomenae (5) 28-31

Cyprinodontidae Lebistes reticulatus (7) 26-28 Thayeria obliqua (6) 17-19

Poecilidae Xiphophorus helleri (8) 47-50 Cichlidae Aequidens pulcher (9) 40-45 Anabantidae Trichogaster trichopterus (10) 37-39

0.14-0.15 0.10-0.13 0.15-0.17 0.39-0.4 1 0.50-0.54 0.i0-0.12 0.39-0.45 2.33-2.88 2.3C2.60 1.13-1.15

Species 1, 2, 7 and 8 are stomachless. The stomachs of species 3 and 9 were not investigated.

2. Histological Procedure and Autoradiography

Each animal received a single intraperitoneal injection of 1 FCi g-' of body weight of methyl-tritiated thymi- dine (["ITdR), sp. act. 48 Ci mmole-', AMER- SHAM International Ltd., U.K.). Animals were decapitated 1 h after injection. Immediately after sacri- fice the ventral surface was opened and the gut was excised. The alimentary tract was cut at five levels (oesophagus, stomach (when present) and upper, middle and lower intestine) and fixed in Bouin's fluid for 24 h. After dehydration the tissues were embedded in paraffin wax and 4 Fm sections were sliced and mounted on light microscope slides. Radioautographs were prepared using Ilford K5 liquid photoemulsion diluted 1:l with bidistilled water. After an exposure time of 18 days at 4°C in light-tight boxes, the autoradi- ographs were developed according to standard methods (Rogers 1979) and stained with haematoxylin.

The labelling indices (TLI: percentage of cells incor- porating [3H]TdR) were determined in the epithelial cells. One thousand cells of each region were counted in each animal. Since the gut epithelium of some speci- mens was thin, 4-5 sections had to be used to count all cells. Nuclei covered with more than 5 silver grains were regarded as labelled (background: 2-3 grains per nucleus). The numerical data were calculated and graphs were drawn with the help of Statgraphics (stat- istical graphics system; STSC Inc. 1986) software. In order to check for the presence of mucus-secreting

cells, additional slides were stained by the periodic acid-Schiff (PAS) method (McManus and Mowry 1960, Pearse 1968-1970).

Results

General Histology

The general histological appearance of the diges-

tive tract of fishes has been described in the general textbooks of Andrew (1959) and Patt and Patt (1969). As compared to higher ver- tebrates, this structure is relatively simple. Briefly, in our material, the oesophageal mucosa is lined by a stratified epithelium with flattened and cuboidal cells at the lumen and numerous saccular, mucus-secreting, PAS positive cells. The details of the arrangement vary somewhat between different species.' The surface epi- thelium of the stomach (when present) consists of columnar cells with the apical portion strongly stained by PAS. The greater part of the mucosa is occupied by the gastric glands. They possess but one type of cell producing both pepsin and hydrochloric acid. The arrangement of the indi- vidual glands and their degree of branching vary between species. The intestine is lined through- out by a prismatic epithelium composed of brush border cells (absorptive cells) and goblet cells (mucus-secreting cells). Some cells are adjacent to the basal lamina and are interpreted as undif- ferentiated elements. A distinction between the villi and tubular glands from the surface (crypts of Lieberkuhn) is lacking in fishes.

A utoradiography

Labelled cells were observed in all parts of the gut, but examination of radioautographs of sto- machs from all species revealed a lesser number of labelled nuclei than in other parts of the gas- trointestinal tract. For each species the mean TLI and standard error (SEM) of the epithelial cells lining the oesophagus, stomach and upper, middle and lower parts of the intestine are given in Figure 1. Analysis of variance (Table 2)

Cell Prolijkration in the Digestive Mucosa of Fishes 227

13

IS

13

33p 28

I 2 3 4 5 6 7 8 9 10

21r 23

I 2 3 4 5 6 7 8 910

13

-7$ 2 3 4 5 6 7 8 9 10

Species Species Species

Fig. 1. TLI of lining epithelium cells of oesophagus (a), stomach (b) and upper (c), middle (d) and lower (e) intestine of fishes killed 1 h after a single [.'H]TdR exposure. The numbers refer to the species listed in Table 1.

revealed significant differences among the lumi- nal epithelia with respect to the level of the intestinal tract. The upper intestine epithelium had a higher TLI than that of other digestive levels. If columnar cell nuclei were covered with silver grains, goblet reactive cells were not observed.

Discussion

The widely accepted method for detecting cells engaged in DNA synthesis in vivo is by their uptake of ["ITdR, identified using autoradiog- raphy. The thymidine labelling index (TLI) rep- resents the proportion of cells that have incorpor- ated radiothymidine into DNA during a brief exposure period and it is generally admitted that an increase in the value of this parameter is related to an increase in cell proliferation

(Epifanova 1966, Schultze and Maurer 1973, Cheng and Leblond 1974, Steward et al. 1980, Paridaens et al. 1985, Jost 1986, Jost and Potten 1986, Kiss et al. 1986a,b, Zorn and Carneiro 1987). It should, nevertheless, be borne in mind that an increase in ['HITdR incorporation into DNA and a lack of change in cell multiplication can sometimes be observed (Jozan et al. 1985), and explained by reference to the metabolic pathway of thymidine (Maurer 1981). On the other hand, a difference in the cell kinetic pattern might also explain the increased radiothymidine incorporation into upper intestine nuclei without subsequent increase in cell proliferation. Indeed, an increased number of cells entering the S phase, but with a slow DNA synthesis, may explain both the increased incorporation and the unchanged cell number. Whether or not our results may be explained by this mechanism can- not be deduced from our study.

Many investigators have studied the proliferat-

228 Robert Kiss et al.

Table 2. Mean labelling index (TLI) in different parts of the gut mucosa

Number of species Mean TLI measured (YO 2 SEM)

Oesophagus (ES) 10 2.69 2 0.63 Stomach (ST) 4 0.92 2 0.25

Middle intestine (M.int) 10 4.46 2 0.96 Lower intestine (L.int) 10 1.46 t 0.36

Upper intestine (U.int) 10 8.00 2 1.16

Statisrical significances

ES vs. St ES vs. U.int ES vs. M.int ES vs. L.int St vs. U.int St vs. M.int St vs. L.int U.int vs. M.int U.int vs. L.int M.int vs. L.int

Ns ( P > 0.05) P < 0.001 P < 0.05 P < 0.05 P < 0.01 P < 0.05 Ns P < 0.01 P < 0.001 P < 0.001

ive activity of gut epithelia in mammals (see Introduction), but few studies have been conduc- ted on the fish digestive mucosa (Hyodo 1965, Hyodo-Taguchi 1970. Garcia and Johnson 1972, Gas and Noaillac-Depeyre 1974).

The epithelium lining the digestive tract of fishes shares some interesting kinetic features with that of mammals. The upper and middle parts of the intestine have the highest proliferat- ive index, the oesophagus, the stomach and the lower intestine the lowest. If our data are directly comparable to those published on mammals, it should be noted that the labelling indices are much weaker than those reported for murines (Schultze et al. 1972, Kovacs and Potten 1973); this might be related to the weak intensity of fish metabolism. In the fundic mucosa. cell prolifer- ation takes place in the region of the isthmus between the gastric epithelium and the glands. a situation quite similar to that reported in rodents (Hattori and Fujita 1976).

After [3H]TdR flash labelling there is greater DNA synthesis activity in the mid and basal portions of intestine villi folds in all species inves- tigated and, like Gas and Noaillac-Depeyre (1974), we are unable to describe a definite zone of proliferation. This is not surprising if we remember that fishes (like amphibians and rep- tiles) do not possess the tubular invaginations from the surface epithelium (crypts of Lieber- kuhn) which are a characteristic of higher ver- tebrates and the starting point of all renewal phenomena. On the other hand, our obser-

vations disagree with those of Vickers (1962), who described a definite zone of proliferation in the goldfish intestine. As shown in Table 2, there is a significant difference in the mean labelling indices between the lower intestine region and the other two regions, the TLI in the former being weaker. The existence of a proximal-distal gradient has been observed in mammals (Kovacs and Potten 1973) as well as in lower vertebrates (Wurth and Musacchia 1964, Gas and Noaillac- Depeyre 1974). In contrast, no such observation was reported by Stroband and Debets (1978) in the grasscarp.

Interestingly, no species differences in diges- tive mucosa cell proliferation were detected. This may be due to the wide range of individual vari- ation, which is not satisfactorily explained. Therefore, a real absence of differences among species cannot be ruled out.

Careful examination of sections revealed the presence of silver grains on the nuclei of absorp- tive cells, suggesting that functional mature cells might be able to proliferate. A similar obser- vation was also reported by Stroband and Debets (1978). New differentiated cells can be produced during adult life in two ways: by mitotic division of existing differentiated cells giving pairs of daughter cells or the same type; by differen- tiation of undifferentiated stem cells. From our material it seems that both ways are used in the fish intestinal epithelium. This is in contrast to the mammalian intestine. where only undifferen- tiated cells are able to proliferate (Lipkin 1973. Cheng and Leblond 1974). However, a pro- longation of the proliferation ability of fish intes- tinal cells cannot be ruled out. Further autoradio- graphic and ultrastructural examinations of proliferating cells are required before any defini- tive conclusion can be drawn.

Stomachless species were included in our study and no differences in the intestine could be observed between these and fishes with a sto- mach. It has been postulated that the absence of a stomach might influence protein digestion and absorption of macromolecules in the intestine (Stroband and Kroon 1981). Therefore, we expected the absence of a stomach to be reflected by particularities in the cytophysiology, i.e. cell proliferation of the intestinal mucosa. This was not shown by our results, but the number of species investigated is small and further studies must be conducted before any comprehensive conclusion can be drawn. . To summarize, the findings with tritiated thy- midine allow the following conclusions. The digestive mucosae of the 10 investigated species of fish have a cell renewal system comparable to that of the intestine in goldfish (Vickers 1962, Hyodo 1965), carp (Gas and Noaillac-Depeyre

Cell Proliferation in the Digestive Mucosa of Fishes 229

1974) and grasscarp (Stroband and Debets 1978). For a given region no differences were detected between species belonging to various families. T h e upper intestine has the highest proliferation activity and its apparently functional cells are still able t o enter into the S phase of the cell cycle.

References

A/-Dewachi, H. S., Wright, N . A , , Appleton, D. R. , Watson, A . J . 1975. Cell population kinetics in the mouse jejunal crypt. Virchows Arch. Cell Path.

Andrew, W . 1959. Textbook of Comparative Histology. Oxford University Press, New York.

Bertalanffy, K. P., Nagy, K. P. 1961. Mitotic activity and renewal rate of the epithelium cells of human duodenum. Acta anal. 45, 362-370.

Bleiberg, H., Buyse, M., Galand, P. 1985. Cell kinetic indicators of premalignant stages of colorectal can- cer. Cancer 56, 124-129.

Bleiberg, H., Mainguer, P., Vandenhende, J. , Galand, P. 1971. Mesure autoradiographique de la proliftr- ation cellulaire a differents niveaux du tractus dige- stif normal et pathologique. Utilisation de biopsies incubtes in vitro. Revue eur. Etud. clin. biol. 16.

Cairnie, A . B., Lamerron, L . F., Steel, G. G. 1965. Cell proliferation studies in the intestinal epithelium of the rat. I. Determination of the kinetic par- ameters. Exp. Cell Res. 39, 52&538.

Cameron, I. L . , Greulich, R. C. 1963. Evidence for an essentially constant duration of DNA synthesis in renewing epithelia of the adult mouse. J . Cell Biol.

Cheng, H . , Bjerknes, M. 1982. Whole population cell kinetics of mouse duodenal, jejunal, ileal and colonic epithelia as determined by autoradiogra- phy and flow cytometry. Anat. Rec. 203,. 251-264.

Cheng, H. , Leblond, C. P. 1974. Origin, differen- tiation and renewal of the four main epithelial cell types in the mouse small intestine. V. Unitarian theory of the origin of the four epithelial cell types. Am. J . Anat. 141. 537-562.

Chiakulas, J . J., Scheving, L. E. 1961. The cyclic nature and magnitude of cell division in gastric mucosa of Urodele larvae reared in the pond and laboratory. Biol. Bull. mar. biol. Lab, Woods Hole

Cleaver, J . E. 1967. Thymidine metabolism and cell kinetics. Monographs iii Frontiers of Biology. Vol. 6. North Holland, Amsterdam.

Dormer, P., Moiler, E. D. 1968. Autoradiography of the non-uniformity of cell kinetics as revealed in the forestomach of the mouse. Exp. Cell Res. 49,

Epifanova, 0. I . 1966. Mitotic cycles in estrogen- treated mice: a radioautographic study. Exp. Cell res. 42, 562-577.

Garcia, M. N. , Johnson, A. 1972. Cell proliferation kinetics in goldfish acclimated to various tempera- tures. Cell Tissue Kinet. 5. 331-339.

18, 225-242.

233-239.

18, 31-39.

120. 1-7.

495-503.

Gas, N., Noaillac-Depeyre, J . 1974. Renouvellement de I’tpithClium intestinal de la carpe (Cyprinus carpi0 L.). Influence de la saison. C.r. hebd. Skanc. Acad. Sci., Paris 2790, 1085-1088.

Hattori, T . , Fujita, S. 1976. Tritiated thymidine autora- diographic study of cell migration and renewal in the pyloric mucosa of golden hamster. Cell Tissue Res. 175, 49-57.

Hughes, W . L., Bond, V . P., Brechter, G., Cronkite, E. P., Painter, R. B., Quastler, H., Sherman, F. G. 1958. Cellular proliferation in the mouse as revealed by autoradiography with tritiated thymi- dine. Proc. natn. Acad. Sci. U.S.A. 44, 476-483.

Hyodo, Y. 1965. Development of intestinal damage after X-irradiation and 3H-thymidine incorpor- ation into intestinal epithelial cells of irradiated goldfish, Carassius auratus at different tempera- ture. Radial. Res. 26. 383-394.

Hyodo-Taguchi, Y. 1970. Effect of X-irradiation on DNA synthesis and ell proliferation in the intesti- nal epithelial cells o i: goldfish at different tempera- tures with special reference to recovery process. Radial. Res. 41, 568-578.

Jost, S. P. 1986. Renewal of normal urothelium in adult mice. Virchows Arch. Cell Path. 51, 65-70.

Jost, S. P., Potten, C. S. 1986. Urothelial proliferation in growing mice. Cell Tissue Kinet. 19, 155-160.

Jozan, S., Gay, S., Marques, B., Mirouze, A , , David, J . F. 1985. Oestradiol is effective in stimulating 3H-thymidine incorporation but not on prolifer- ation of breast cancer cultured cells. Cell Tissue Kinet. 18, 457-464.

Kiss, R . , Danguy, A. , Heuson, J . C . , Paridaens, R . 1986a. Effect of estradiol on proliferation and on cell loss in the MXT mouse mammary tumor. Anticancer Res. 6, 1321-1328.

Kiss, R., de Launoit, Y., Lenglet, G . , Danguy, A . 1986b. Evaluation on the cell cycle kinetic par- ameters in the mucosal epithelium of the amphib- ian digestive tract. Archs Biol., Brux. 97, 237-257.

Kovacs, L. Potten, C. S. 1973. An estimation of pro- liferative population size in stomach, jejunum and colon of DBA-2 mice. Cell Tissue Kinet. 6 ,

Leblond, C. P., Messier, B. 1958. Renewal of chief cells and goblet cells in the small intestine as shown by radioautography after injection of 3H-thymi- dine into mice. Anat. Rec. 132, 247-259.

Lennartz, K. J. , Maurer, M. 1964. Autoradiograph- ische Bestimmung der Dauer der DNS-Verdop- plung und der Generationzeit beim Ehrlich Ascites Tumor der Maus durch Doppelmarkierung mit 14C- und 3H-Thymidine. Z. Zellforsch. 63, 478-495.

Lipkin, M. 1973. Proliferation and differentiation of gastrointestinal cells. Physiol. Rev. 53, 891-915.

Lipkin, M., Quastler, H. 1962. Cell population kinetics in the colon of the mouse. J . clin. Invest. 41,

McAvoy, J. W., Dixon, K. E. 1977. Cell proliferation and renewal in the small intestinal epithelium of metamorphosing and adult Xenopus laevis. J . exp. Zool. 202, 129-138.

McManus, J . F. A , , Mowry, R. W . 1960. Staining Methods. Histologic and Histochemical. P. B. Hoeber, Inc., New York.

125-134.

14 1- 146.

230 Robert Kiss et al.

Maurer, H. R. 1981. Potential pitfalls of (3H)-thymi- dine techniques to measure cell proliferation. Cell Tissue Kinet. 14, 111-120.

O'Steen, W . K., Walker, B. E. 1960. Radioautographic studies of regeneration in the common newt. 1. Physiological regeneration. Anat. Rec. 137,

Paridaens, R., Danguy, A , , Leclercq, G., Kiss, R., Heuson, J . C. 1985. Effect of castration and estra- diol pulse on cell proliferation in the uterus and the MXT mouse mammary tumor. J . natn. Cancer Inst. 74, 1239-1246.

Patt, D. I . , Patt, G. R. 1969. Comparative Vertebrate Histology. Harper and Row, New York.

Patten, S. F. Jr. 1960. Renewal of the intestinal epi- thelium of the Urodele. Exp. Cell Res. 20, 638-641.

Pearse, A . G. E. 1968-1970. Histochemistry. Theoreti- cal and Applied, 2 Vol. J. R. A. Churchill, Lon- don.

Pilgrim, C. 1964. Autoradiographische Bestimmung der Dauer der DNS-Verdopplung bei Verschied- enen Zellarten von Maus und Ratte nach einer neunen Doppelmarkierungmethode. Anat. Anz . 115, 12U-133.

Rogers, A . W . 1979. Techniques of Autoradiography, 3rd Edn. Elsevier-North Holland, New York.

Schultze, B., Maurer, W . 1972. Proliferation of liver epithelium in three week-old rats. Cell Tissue Kinet. 6 , 477-482.

50 1-509.

Schultze, B., Haack, V . , Schmeer, A. C., Maurer, W . 1972. Autoradiographic investigation on the cell kinetics of crypt epithelium in the jejunum of the mouse. Cell Tissue Kinet. 5 , 131-145.

Stewart, F. A., Denekamp, J. , Hirst, D. G. 1980. Proliferation kinetics of the mouse bladder after irradiation. Cell Tissue Kinet. 13, 75-89.

Stroband, H. W . J., Debets, F. M. H. 1978. The ultra- structure and renewal of the intestinal epithelium of the juvenile grasscarp, Ctenopharyngodon idella (Val.). Cell Tissue Res. 187, 181-200.

Stroband, H. W . J . , Kroon, A. G. 1981. The develop- ment of the stomach in Clarias lazera and the intestinal absorption of protein macromolecules. Cell Tissue Res. 215, 397-415.

Vickers, T. 1962. A study of the intestinal epithelium o f the goldfish Carassius auratus: its normal struc- ture, the dynamics of cell replacement and the changes induced by salts of cobalt and manganese. Q. J . microsc. Sci. 103, 93-110.

Willems, M. A., Galand, P. 1970. Etude de la prolifer- ation cellulaire au niveau des muqueuses fundiques et rectales chez le chien par la methode du double marquage in virro. Archs Biol., Brux. 81,427-440.

Wurth, M. A . , Musacchia, X . J . 1964. Renewal of intestinal epithelium in freshwater turtle, Chry- semis picta. Anat. Rec. 148, 427-439.

Zorn, T. M. T., Carneiro, J . 1987. Cell proliferation in synovial membrane of young mice. Acta Anat. 128, 1%22.