Observations on Intranuclear Crystal and Nucleolar Size at Different Stages of Cell Differentiation in the Midgut Epithelium of Several Insects

y. Cell Sci. 22, 87-97 (1976) 87 Printed in Great Britain

OBSERVATIONS ON INTRANUCLEAR CRYSTAL AND NUCLEOLAR SIZE AT DIFFERENT STAGES OF CELL DIFFERENTIATION IN THE MIDGUT EPITHELIUM OF SEVERAL INSECTS

J. GOURANTON AND D. THOMAS Groupe de Reclierches de Biologie Cellulaire, Avenue du General Leclerc, 35031 Rennes Cedex, France

SUMMARY Based on an inverse size relationship between nuclear crystal and nucleolus in different cells it has been postulated by several authors that the crystal develops from nucleolar materials. The purpose of the present paper is to investigate the validity of this argument. Intranuclear proteinaceous crystals appear in differentiating midgut cells of Gyrinus marinus and Tenebrio molitor. In an autoradiographic study we have previously demonstrated in these two species that the crystals do not develop from nucleolar materials. However, an inverse relationship with regard to size is observed between these 2 structures during the cell differentiation: the cross- sectional area of the nucleolus decreases when the cross-sectional area of the crystal increases. But a decrease in size of the nucleolus is also observed during the differentiation of the midgut cells of Gyrinus natator where the crystals are not present. Consequently an inverse size relationship cannot be a sufficient argument to postulate that intranuclear crystals and nucleoli are interconvertible structures; decrease in size of the nucleolus is not related to development of the intranuclear crystal.

INTRODUCTION The nuclear inclusions which have been described in numerous cells are quite diverse and several types have been recognized. In the present paper only intranuclear proteinaceous crystals will be considered. Such crystals have been regularly observed in a variety of plant and animal cells. The origin of these crystals has been a subject of interest for all the authors who described them. A close association of the intranuclear crystal with the nucleolus has not been frequently observed (Wergin, Gruber & Newcomb, 1970; Le Moigne & Monnot- Sauzin, 1971; Coleman & Phillips, 1972; Unzelman & Healey, 1972) and some authors (Unzclman & Healey, 1972) suggested that this association might be expected purely on the basis of sizes of the two structures. Several authors have observed a reciprocal relationship between the nucleolus and the intranuclear crystal in that the size of the nucleolus decreases while the crystal size increases. This inverse relationship, first mentioned by some authors at the beginning of this century (see review in Wergin et al. 1970) was observed by Saurer (1962) with the protein crystals in the nucleus of glandular cells of Pinguicula caudata. A similar 88 J. Gouranton and D. Thomas relationship is described by Wergin et al. (1970) in differentiating leaf mesophyll cells of Camplyoneuron phyllitidis. Unzelman & Healey (1972), in a study on the nuclear crystals in the trichomes of Pharbitis nil, reported that, concomitant with crystal growth, nucleoli undergo a 50 % decrease in cross-sectional area. In the young fronds of Asplenium fontanum, Arsanto (1973) states that the crystals increase in size while the nucleoli become smaller. It does not appear that this relationship always exists, for the observations of Fabbri & Menicanti (1970) in Blechnum brasiliense do not agree with the preceding studies. These authors did not find size interdependence between nucleolus and crystals in the sense stated above. Moreover, they observed an increase in nucleolus size at the same time as the volume of the crystals increased. The inverse relationship between the sizes of the intranuclear crystals and nucleoli has been considered by some authors as an argument to suggest the origin or the development of crystals from nucleolar materials (Arsanto, 1973; see also review in Wergin et al. 1970). Our intention, in the present work, is to investigate the validity of this argument. We thought that it would be interesting to compare the sizes of the nucleoli with the sizes of the crystals in cells where it has been demonstrated that the nucleoli do not play a direct part in the crystal synthesis. We have shown, using autoradiography, that the crystal proteins of the midgut cells of two insects, Tenebrio molitor (Thomas & Gouranton, 19730) and Gyrinus marinus (Gouranton & Thomas, 1974), are synthe- sized in the cytoplasm and then migrate to the nucleus where they crystallize. In order to make further comparisons with the results obtained for these two insects, we thought that measurements of nucleolar sizes, during cell differentiation, should also be taken in the midgut epithelial cells of an insect where no intranuclear crystals are seen. Gyrinus natator is related to Gyrinus marinus and carries no crystals in the nuclei of midgut cells (Gouranton, 1972).

MATERIALS AND METHODS Our observations on crystals were made in the midgut cells of 2 adult coleopterons: Gyrinus marinus Gyll. and Tenebrio molitor L. The intranuclear crystals are never found in the embryonic cells but appear and increase as the cells become differentiated. As already mentioned, midgut cells of Gyrinus natator L., which are devoid of intranuclear crystals, were also examined. In the midgut of these 3 adult coleopterons the replacement cells form crypts which project like villi on the outer surface of the midgut. The new cells are obtained by division of the embryonic cells at the extremity of the crypt and then migrate to renew the midgut epithelium (Fig. 1). In the midguts of G. marinus and G. natator measurements were taken by light microscopy of semithin sections of material included in an Araldite-Epon mixture. Longitudinal sections of villi of 4 G. marinus and 2 G. natator were photographed; 5 zones were distinguished in the villi from their extremity to the lining of the midgut (Fig. 1). For each insect, from 8 to 34 nuclei of each zone were examined on micrographs at a final magnification of 960; the areas of the nuclei, nucleoli and intranuclear crystals (when present) were cut out and weighed to measure them. In order to have comparable results, in each zone, the weights were used to calculate ratios of the cross-sectional areas of the nucleoli or the crystals to the cross-sectional areas of the nuclei. The results are given for each insect separately. We thought that it would be interesting to have measurements on electron micrographs for Intranuclear crystals and nucleolar size 89 one of the two insects with crystals. In the midgut of T. violitor the cross-sectional sections of the nucleoli, crystals and nuclei were evaluated on electron micrographs. Since it appeared difficult, in electron microscopy, to distinguish the different zones along the crypts, the stage of differentiation of the cells was evaluated using autoradiography after an injection of tritiated thymidine. We have used and previously described this method to investigate the rate of formation of the crystals in the midgut of T. molitor (Thomas & Gouranton, 19736). A few hours after a tritiated thymidine injection, only the replacement cells of the extremity of the crypt are labelled. If the insects are kept alive longer, the labelled replacement cells migrate progressively. In the present work, measurements were taken on the labelled nuclei, which were the nearest to the lining of the midgut, in insects killed 6, 30, 48 or 96 h after injection of the DNA precursor. These times represent therefore the ages of the differentiating cells in the midgut. For each time, from 19 to 116 nuclei were examined in several insects. The cross- sectioned areas were measured as already mentioned for light-microscopical micrographs. For the mean values at each time the standard errors of the means were calculated.

Multiplication of replacement cells

Fig. 1. Diagram to illustrate a regenerative crypt in midgut of the adult coleopterons, G. marinns and T. molitor. New cells are produced by division of the embryonic cells at the extremity and they then migrate to renew the midgut epithelium. The 5 zones represent successive stages in midgut cell differentiation. The position of the section in Fig. 3 is shown by a dotted outline.

RESULTS Observations in G. marinus Close physical association between the nucleolus and intranuclear proteinaceous crystals was sometimes, but rarely, observed in the midgut cells. When this association was noted, generally one only of the intranuclear crystals was associated with the nucleolus (Fig. 2). At the extremity of the regenerative crypt the embryonic cells actively divide and produce new cells which migrate progressively to the lining of the midgut as the old epithelial cells are eliminated in the gut lumen. Therefore the 5 zones which are 9$ jf. Gouranton and D. Thomas Intranuclear crystals and nucleolar size 91 distinguished along the crypt drawn in Fig. 1 represent 5 successive stages in the midgut-cell differentiation. The crystals, which are not present in young cells (zones I and II), appear in zone III and then increase in size in zones IV and V. The appearance of the first crystals in a crypt can be seen on the micrograph of Fig. 3. The position of this micrograph is indicated by a dotted outline on Fig. 1.

Table 1. Area ratios nucleolus/nucleus (Nu/N) and crystal/nucleus (CrjN) at different stages of midgut cell differentiation in G. marinus

Insect A Insect B

No. of No. of nuclei nuclei Zone examined Nu/N Cr/N examined Nu/N Cr/N

I 16 0-18 — 20 O-2I — II 31 023 — IS O-22 — III 19 o-ii 013 11 0-09 008 IV 18 006 0-27 12 OO9 o-io V 20 0-07 031 12 0-08 0-16 Insect c Insect D A A I 12 0-19 — 14 0-17 — II 12 0-22 — 21 0-17 — III 13 O-l6 008 12 o-io 007 IV 9 O-II O-II 9 009 013 V 12 0-08 0-17 12 006 018

The mean values of the ratios between the cross-sectional areas of the nucleoli or the crystals and the cross-sectional areas of the nuclei are given in Table 1 for each zone, and separately for the 4 insects studied. The evolution of the ratios is shown on the curves of Fig. 4. It clearly appears, in all insects studied, that the area ratio of nucleolus to nucleus decreases when the area ratio of crystals to nucleus increases.

Observations in G. natator The regenerative crypts are quite similar to those of G. marinus but no crystals are observed. As above, the ratios between the cross-sectional areas of the nucleoli and the nuclei were calculated. The results are given in Table 2 for each zone and separately for the 2 insects studied. The curves of Fig. 5 show the evolution of this ratio.

Fig. 2. Electron micrograph showing intranuclear crystals (cr) one of which is associated with the nucleolus (nu) in a midgut cell of G. marinus. x 25000. Fig. 3. Light micrograph of portion of a crypt in G. marinus, where the crystals (cr) appear in the nuclei (in the middle of the micrograph). Then they increase in size (to the right). The position of this micrograph is outlined in Fig. 1. x 960. J. Gouranton and D. Thomas

0 30 0-30 -

o 020 8 0-20 5.

0-10 0 10

002 - 002 - III IV V I II III IV V Zones Zones

1 J c D 0-30 0-30

•

2 0-20 9 020 n P •—\ > \ \ ft'

0-10 - 010 0'' cr

002 002 I 1 I 1 1 • i i —J *~ IV V IV V Zones

Fig. 4. Evolution of the cross-sectional area ratios Nu/N (•) and Cr/N (O) during differentiation of midgut cells of 4 individual G. marinus (A, B, C, D).

Table 2. Area ratios nucleolusj nucleus (Nu/N) at different stages of midgut cell differentiation in G. natator

Insect E Insect F No. of No. of nuclei nuclei /One examined Nu/N examined Nu/N

I 34 0-22 24 0-15 II 23 0-29 18 O-2I III 24 O-24 OI5 IV IS OI3 16 o-ii V 12 O-OQ 8 o-io Intranuclear crystals and nucleolar size 93 Though intranuclear crystals are lacking in G. natator the evolution of the area ratio nucleolus/nucleus is comparable to that observed in G. marinus. It decreases from zone II to zone V.

Observations in T. molitor For T. molitor the measurements were taken on electron micrographs and a new method was used to distinguish the stages of cells differentiation. The age of the midgut cells was evaluated using autoradiography after tritiated thymidine injections.

0-30 0-30

o 0-20 o 0-20

0-10 0-10

002 - 002 - IV IV Zones Zones Fig. 5. Evolution of cross-sectional area ratios Nu/N during differentiation of midgut cells of 2 individual G. natator (E, F).

Table 3. Area ratios nucleolus/nucleus (Nu/N) and crystal/nucleus (Cr/N) at different stages of midgut cell differentiation in T. molitor

Age of differentiating No. of nuclei cell, h examined Nu/N Cr/N 6 116 0-093 + 0-005 3° 57 0-106 ±O-OII 48 19 0-096 ±0-009 O-O2I ±O-OO2 96 25 0-060 ± 0-006 0-050 ±O-OO7 Values of Nu/N and Cr/N are stated as means ± S.E.M.

The area ratios nucleolus/nucleus and crystal/nucleus are given in Table 3. Intra- nuclear crystals are not present in young cells (Figs. 6, 7). They appear in cells after 48 h of differentiation (Fig. 8) and then increase in size (Fig. 9). The evolution of area ratios between the nucleolus or the crystals and the nucleus is shown by the curves of Fig. 10. It clearly appears, as in G. marinus, that the area ratio of nucleolus/nucleus decreases when the area ratio crystal/nucleus increases. J. Gouranton and D. Thomas

nu • V

.-*• •'

fe r? •v • Intranuclear crystals and nucleolar size 95

0-125

0-100

0-075 o 5.

0 050

0-025 Y

30 48 96 Time, h Fig. io. Evolution of cross-sectional area ratios Nu/N (•) and Cr/N (O) during differentiation of midgut cells in T. molitor.

Figs. 6-9. Electron micrographs of midgut cells in T. molitor. The ages of the differentiating cells were evaluated using autoradiography after [3H]thymidine injections, x 8400. Fig. 6. 6 h of differentiation: no crystals, nu, nucleolus. Fig. 7. 30 h of differentiation: the cross-sectional area of the nucleolus is important but there is still no crystal observed. (For this autoradiograph a phenidon-containing developer was used, so the grains appear different from those in the other autoradio- graphs.) Fig. 8. 48 h of differentiation: a small intranuclear crystal (cr) is observed. Fig. 9. 96 h of differentiation: the intranuclear crystal is larger. 96 J. Gouranton and D. Thomas

DISCUSSION AND CONCLUSION In the present work we have used different methods to assess the stages of differentiation of cells. These were distinguished according to their positions along the regenerating crypts in Gyrinus midgut and using autoradiography in Tenebrio midgut. The measurements have been taken on light micrographs for Gyrinus and on electron micrographs for Tenebrio. The results were given separately for each insect {Gyrinus) or not {Tenebrio). Whichever method was used, the results were similar for the insects studied. In the midgut cells of G. marinus and T. molitor the intranuclear crystals appear during cell differentiation and then increase in size. The cross-sectional areas of the nucleolus rapidly increases at the beginning of the cell differentiation and then decreases. Since the cross-sectional area of the nucleus progressively increases during cell differentiation it appeared more interesting to have the results in ratios between the area of the nucleolus or the crystal and that of the nucleus. For the midgut cells of G. marinus and T. molitor our results are in agreement with those of authors who described a reciprocal relationship between nucleolus and intranuclear crystal sizes. Using autoradiography we have previously investigated the kinetics of labelling of the crystals in the midgut cells of these 2 insects and demonstrated the intracytoplasmic site of synthesis (Thomas & Gouranton, 1973 a; Gouran- ton & Thomas, 1974). That we rarely observed a close physical association between some intranuclear crystals and the nucleoli, is consistent with these results. The crystals do not form from nucleolar materials but we have, however, observed an inverse relationship between the two structures. The evolution of nucleolar size is quite similar in midgut cells of G. natator though the crystals are not present in this insect. Consequently it may be concluded that an inverse size relationship cannot be sufficient argument to postulate that intranuclear crystals and nucleoli are interconvertible structures and that the decrease in size of the nucleolus is not related to the development of the intranuclear crystal.

The authors would like to thank A. Cavalier, F. de Sallier Dupin, M. Mathelier and B. Morille for technical assistance.

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GOURANTON, J. & THOMAS, D. (1974). Cytochemical, ultrastructural, and autoradiographic study of the intranuclear crystals in the midgut cells of Gyrinus marinus Gyll. J. Ultrastruct. Res. 48, 227-241. LE MOIGNE, A. & MONNOT-SAUZIN, M. J. (1971). Etude au microscope electronique d'inclusions nucleaires chez des planaires (Turbellaries, Triclades). J. Microscopie 10, 107-112. SAURER, W. (1962). Diploma thesis ETH Zurich quoted in Ultrastructural Plant Cytology by A. Frey-Wyssling & K. L. Miihlethaler. Amsterdam: Elsevier, 1965. THOMAS, D. & GOURANTON, J. (1973a). Etude, au moyen de precurseurs marques, de la synthese de cristaux proteiques intranucleaires chez Tenebrio molitor L. J. Microscopie 16, 287-298. THOMAS, D. & GOURANTON, J. (19736). Duree de formation des cristaux proteiques intranucleaires de l'intestin moyen de Tenebrio molitor. J. Insect Physiol. 19, 515-522. UNZELMAN, J. M. & HEALEY, P. L. (1972). Development and histochemistry of nuclear crystals in the secretory trichome of Pharbitis nil. J. Ultrastruct. Res. 39, 301-309. WERGIN, W. P., GRUBER, P. J. & NEWCOMB, E. H. (1970). Fine structural investigation of nuclear inclusions in plants. J. Ultrastruct. Res. 30, 533-557.

(Received 12 March 1976)