Occurrence of apoptosis in serosa of Periplaneta americana l. (Blattaria: blattidae): ultrastructural and biochemical features

Pergamon PII: S0022-1910(97)00075-9

J. Insect Physiol. Vol. 43, No. 11, pp. 999–1008, 1997  1997 Elsevier Science Ltd All rights reserved. Printed in Great Britain 0022-1910/97 $17.00 + 0.00

Occurrence of Apoptosis in Serosa of Periplaneta americana L. (Blattaria: Blattidae): Ultrastructural and Biochemical Features SERGIO BARNI,*† SIMONETTA LAMBIASE,* ALDO GRIGOLO,* LUCIANO SACCHI,* SILVIA CORONA,* ANNA IVANA SCOVASSI,‡ UGO LAUDANI* Received 2 April 1997; revised 27 May 1997

In 16–17-day-old embryos of Periplaneta americana, the amnion-serosa penetrates the cavity of the middle intestine, where it forms a cluster of compressed roundish cells. We demonstrated that these cells degenerate throughout apoptosis. The programmed cell death revealed by morphological and biochemical approaches showed all the apoptotic steps: chromatin fragmentation and pyknosis, cytoplasm condensation, karyorrhexis, cytoplasm cleavage. Nevertheless, some ultrastructural peculiarities (atypical heterochromatin arrangement, appearance of nuclear envelope protrusions, absence of nucleolar structures) suggest that the apoptotic expression partially depends on the biological situation (type of organism and inducing factors) in which the programmed cell death takes place. The presence of histiocytic cells internalizing cell debris, of apoptotic and non-apototic derivation, may be correlated with the importance of recycling substances useful for embryo growth.  1997 Elsevier Science Ltd. All rights reserved Periplaneta americana Apoptosis Serosa

Embryogenesis Dorsal organ

INTRODUCTION

As early as 1889, Wheeler, in describing the embryonic development of Blatta (syn. Blattella) germanica, had demonstrated in a dorsal portion of the embryo a transitory structure, that he called ‘dorsal organ’, composed of serosa cells and probably of amniotic cells. This structure, originally extra-embryonal, while invaginating inside the embryo immediately before the dorsal closure of the thoracic sclerites, presented a degeneration of the basal nuclei the nature of which obviously could not be clarified at that time. Subsequently, such a dorsal organ was described in Orthoptera (Heinig, 1967), in Hemiptera (Enslee and Riddiford, 1981) and in Diptera (Abbassy et al., 1995), which suggests that it is present in other orders of pterygote insects. In Apterygota a dorsal organ analogous to that of Pterygota was demonstrated by Uzel, 1897a, b, 1898; Tiegs

1944; Jura 1972. These authors attributed secretory functions to it. This organ, however, does not appear homologous with that of the pterygote insects, since it forms before the development of the germinal band. Continuing our research aimed at clarifying the embryologic aspects of endosymbiosis in Blattaria (Laudani et al., 1995; Sacchi et al., 1996; Lambiase et al., 1997), we have recently observed in embryo of Periplaneta americana the presence of a dorsal organ, homologous to that described by Wheeler (1889), of which the cells, derived from the serosa and amnion, undergo degenerative processes. In this study carried out on Periplaneta americana we analyzed, by ultrastructural and biochemical approaches, the mechanisms involved in the cell degeneration of the dorsal organ and we correlated it with the ‘orthodox’ event of apoptosis so far described in vertebrates and invertebrates. MATERIALS AND METHODS

*Dipartimento di Biologia Animale, Universita` di Pavia, Piazza Botta 9, 27100, Pavia, Italia. †To whom all correspondence should be addressed. Fax: + 39-382506290 E-mail: [email protected]. ‡Istituto di Genetica Biochimica ed Evoluzionistica, C.N.R., Via Abbiategrasso 207, 27100, Pavia, Italia.

Experimental insects Specimens of Periplaneta americana (Blattaria: Blattidae) were reared in a controlled environment at 26°C at about 65% relative humidity with a natural regi-

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men of light and darkness. The insects were raised on a mixture of fresh lettuce and bread crumbs, enriched with dog food, and received water ad libitum. We experimented on 16–17-day-old embryos obtained from oothecae dated from the time when they were deposited. Dehiscent oothecae (Lenoir-Rousseaux and Lender, 1970) were opened in Yeager’s solution (Yeager, 1939) starting from the sutural opening of the keel. Eggs were treated with sodium hypochlorite to obtain dechorionisation, then washed twice in Yeager’s solution in order to interrupt the action of the first step of the treatment. Finally, the vitelline membrane was always mechanically removed in ice cold Yeager’s solution. Light microscopy Embryos were fixed in methanol:acetic acid (3:1) for 5–6 days at 4°C, washed in absolute ethanol for a few minutes and embedded in methacrylate ester. Sections 5 ␮m thick were stained with toluidine blue. Transmission electron microscopy Embryos were fixed for 2 h with 2.5% glutaraldehyde solution in 0.1 M cacodylate buffer (pH 7.2) at 4°C, postfixed for 1.5 h with 1% OsO4 in the same buffer (pH 7.2) at 4°C, dehydrated in graded ethanol solutions and embedded in Epon 812. Preliminarily, semi-thin sections (0.5 ␮m thick) were stained with 1% borated methylene blue and examined at light microscopy. Thin sections (about 600 Å thick) were doubly stained with uranyl acetate in 50% acetone and Reynolds’ lead citrate solution (Reynolds, 1963). The specimens were examined with a Zeiss TEM 900 at 50 kV. DNA gel electrophoresis Ontogeny of the dorsal organ was controlled during the whole embryonic development of the insect (our unpublished findings) so that these cell clusters were collected when they were almost completely located inside the embryos immediately after their migration between the anterior and the middle intestine. In this stage the dorsal organ was detectable externally as a little opalescent body between head and thorax. The dorsal organ from each embryo was removed to a cooled watch-glass in Yeager’s solution. The samples were stored in an Eppendorf tube dipped in dry-ice, and this was frozen in liquid N2 until the electrophoretic assay. To analyze internucleosomal DNA fragmentation, cell clusters from the dorsal organ of 200 seventeen-day-old embryos were tested for each electrophoretic lane. Genomic DNA was rapidly extracted and analyzed by electrophoresis on a 1.8% agarose gel by a procedure recently reported by Herrmann et al. (1994) with minor modifications (Bernardi et al., 1995). RNase A, RNase T1, proteinase K and lambda molecular weight DNA marker VI (from 154 to 2176 bp) were from Boehringer (Mannheim, Germany); agarose for gel electrophoresis was purchased from Pharmacia (Uppsala, Sweden).

FIGURE 1. (a) Sagittal section of the dorsal organ of Periplaneta americana in 16-day-old embryos. (b) Detail of the above: the basal part of the organ presents cells with pyknotic nuclei (arrowhead). (c) Sagittal section of the dorsal organ. The hypodermis of the first thoracic tergite is almost entirely closed over the dorsal organ and degenerating cells can be seen throughout its whole extent. c, cerebrum; do, dorsal organ; hc, hypodermal cells; y, yolk. Bars: 22 ␮m for (a); 11 ␮m for (b); 30 ␮m for (c).

RESULTS

Dating of Periplaneta americana oothecae allowed us to follow the ontogeny of the dorsal organ, which in the embryos at the age of 15 days was situated in the cephalic position, while in those aged 16–17 days the dorsal organ underwent a process of internalization at the level of the first thoracic tergite which, at this point, was not yet closed. At this embryonic stage the dorsal organ

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FIGURE 2. (a) Sagittal section of a 17-day-old embryo showing a mass (arrow) of degenerating cells at the cephalic extremity of the deutoplasmic sac. ai, anterior intestine; e, ectoderm; y, yolk. (b, c) semithin sections in two areas (corresponding to squares of (a)) of the degenerating cell cluster: the cells in contact with the yolk (y) show a roundish shape (early apoptosis) and contain a pyknotic nucleus (arrowheads) surrounded by a basophilic stacked RER (arrows); in the peripheral area beneath ectoderm (c), debris of degenerating cells (arrows) are detectable (late apoptosis). hc, hypodermal cells. Bars: 35 ␮m for (a); 10 ␮m for (b) and (c).

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FIGURE 3. Ultrastructure of the cells localized in the area of the cluster in contact with the yolk. The nuclei are characterized by the presence of electron-dense chromatin organized in roundish bodies, heterogeneous in size, partially in contact with the nuclear envelope (a) or in protruding pockets (b). Presence in the nuclear envelope of protrusions (c, d) containing microvesicles (arrows); in the juxtanuclear region the RER is organized in compressed formations (asterisks). ly, lysosomes. Bars: 1.7 ␮m for (a) and (c); 1.1 ␮m for (b); 0.4 ␮m for (d).

was conical in shape with the vertex towards the cephalic portion, with an average height of about 200 ␮m and an average base diameter of about 150 ␮m. The dorsal organ, having originated from the serosa and probably from amnion cells which bind to this during its retraction

towards the anterior pole of the egg, was in contact with the yolk mass, and its innermost cells penetrated the deutoplasm [Fig. 1(a)]. Its outermost cells, on the other hand, took on the aspect of a simple bathyprismatic epithelium. The nuclei of the deepest cells of this organ had a pyknotic appearance [Fig. 1(b)].

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FIGURE 4. (a, b) Ultrastructure of nuclear segmentation (karyorrhexis) with presence of envelope invaginations (arrows). At the end of the karyorrhexis, the cells contain numerous ‘micronuclei’ (c) of heterogeneous sizes and with different pyknotic chromatin distribution. Bars: 2.5 ␮m for (a) and (b); 1.7 ␮m for (c).

In subsequent phases, the degeneration affected also the outermost cells of the dorsal organ [Fig. 1(c)]. In an advanced phase of the internalizing process, the dorsal organ was almost entirely contained in the yolk sac which was closing over it [Fig. 2(a)]. At the end of this process the first thoracic tergite will complete its own closure. The light microscopy morphology of the degenerating dorsal organ is shown in Fig. 2(a). In the deep cell cluster layers, partially in contact with the yolk mass, the cells showed a polyhedric–roundish morphology, with chromatin and cytoplasm condensation (early apoptosis). The nucleus was characterized by the presence of an intense basophilic chromatin organized in roundish bodies, heterogenous in size [Fig. 2(b)]. At the electron microscopy level this chromatin, that appears very electron-dense, was in contact with a pale nuclear component or underlay the inner membrane of the nuclear envelope [Fig. 3(a, b)]. Osmiophilic clusters

of granular–fibrillar material organized in nucleolar or pseudo-nucleolar formations were never found. In addition, the nuclear envelope was sometimes characterized by the presence of protrusions involving membrane layers and containing microvesicles [Fig. 3(c, d)]. The analysis of the DNA pattern in agarose gel electrophoresis of the degenerating cell cluster showed the typical ladder (Fig. 7) representing the cleavage of chromatin in oligonucleosomes, which often occurs in apoptotic cell death. The cytoplasm, during the nuclear pyknosis, showed a condensation of organelles, and in particular of the rough endoplasmic reticulum [Fig. 3(a, c)], that in light microscopy appeared as intense basophilic formations enclosing the nucleus or organized as myelin figures [Fig. 2(b)]. The presence of lysosomes in the active state (Fig. 3) is indicative of autophagic mechanisms more generally responsible for the proteolytic degradation of cytoplasmic and nuclear components during programmed cell

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FIGURE 5. Ultrastructure of the peripheral cell cluster area. (a) cytoplasm cleavage with formation of cell blebs (arrowheads); (b) apoptotic bodies (arrows); Presence of ‘light’ cell (lc), with histiocytic function, (c) containing numerous dense cell bodies (arrowheads) and (d) myelin figures (arrow) as a result of a partial degradation. Bars: 1.1 ␮m for (a) and (b); 2.5 ␮m for (c); 0.6 ␮m for (d).

death (Machiels et al., 1996). Subsequently, during the progression of cell death, the nuclear envelope underwent invaginations [Fig. 4(a, b)] with the formation of blebs, compartments and then the production of discrete nuclear fragments [Fig. 4(c)]. When karyorrhexis was concluded, the proportion of volume occupied by condensed chromatin varied widely: some micronuclear formations appeared ‘filled’, some others appeared ‘empty’ [Fig. 4(c)]. The peripheral area of the degenerating cell cluster, localized beneath ectoderm, was characterized by the presence of elements in the phase of cytoplasm cleavage [Figs 2(c) and 5(a)] with the formation of apoptotic bodies with a very electron-dense matrix (late apoptosis), many of them containing one nuclear fragment [Fig. 5(b)]. Within cell clusters, most apoptotic bodies were internalized by large, pale and irregularly shaped cells

[Fig. 5(c)], probably of a histiocytic nature. Inside these scavenger cells, the bodies underwent degenerative changes with the formation of multilamellar structures [Fig. 5(d)]. Some ‘intact’ cells produced cytoplasmic processes (herniations), that pocketed into neighbouring scavenger cells, generating numerous intracytoplasmic bodies [Fig. 6(a)], subsequently digested [Fig. 6(b)]. In some respects (low cytoplasm density, presence of lysosomes and yolk platelets), the scavenger cells resembled the vitellophages [Fig. 6(c, d)] lodged within the yolk granules. DISCUSSION

After the observations of Wheeler (1889), the nature of the degeneration of the serosa and amnion cells during the embryogenesis of insects has never been considered. Studies on embryos of Locusta migratoria, carried out

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FIGURE 6. Ultrastructure of two histiocytic cells with amoeboid shape in phase of internalization (a) and degradation (b) of cytoplasmic processes (herniations) derived from neighbouring cells (arrows). In plate b an intracytoplasmic yolk droplet (y) is shown. Two active vitellophages (vt) in contact with the yolk mass (y) and after the internalization of yolk droplets (y) are shown in (c) and (d), respectively. Bars: 0.6 ␮m for (a); 2.5 ␮m for (b); 1.7 ␮m for (c); 1.1 ␮m for (d).

by Pe´tavy (1986) in order to study the contribution of the vitellophages to yolk digestion and cytophagocytosis, have shown that several embryonal and extra-embryonal cells present the same degenerative picture: they undergo a ‘programmed death’, closely ascribable to apoptosis, although the author has interpreted it as ‘involutive necrosis’. Research we have conducted on the behaviour of serosa and amnion cells in some species of Blattaria have shown (unpublished findings) that during the retraction of the serosa-amnion envelope towards the anterior pole of the egg these cells undergo considerable morpho-functional modifications, first assuming the aspect of secretory cells and afterwards showing degeneration of the cytoplasm and the nucleus. It was these observations that induced us to study the phenomenon further and thus permitted, in Periplaneta americana, the analysis of the degenerative aspects of the dorsal organ. At the light microscope level this degeneration is seen to take place in a relatively short period (about two days) of embryonic development, between the 16th and the 17th days, when the embryo has completed the closure of all of its abdominal tergites and of its 2nd and 3rd thoracic tergites, and the middle intestine is wrapped in the yolk lamella, in close contact with the dorsal organ. The morphogenetic phenomena that lead to the union of the anterior intestine with the middle intestine allow the latter to be penetrated by the cells of the dorsal organ. All the degenerative processes that began when the dorsal organ was outside the embryo come to an end in the middle intestine.

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Our morphological and biochemical findings indicate that during the embryogenesis of Periplaneta americana the dorsal organ, derived from the serosa epithelium and probably from amniotic cells, forms a compressed cluster of roundish cells undergoing apoptotic degeneration. The genetic control of physiological cell death (apoptosis) plays an essential role in regulating the cell mass of different structures during embryogenesis, metamorphosis and adult life in both vertebrates and invertebrates. Particularly intense is the apoptotic death during the involution of anatomical structures with a transient usefulness in organisms that undergo metamorphosis (Wyllie et al., 1980; Lockshin, 1981; Robertson and Thomson, 1982; Counis et al., 1989; Compton and Cidlowski, 1992). Apoptosis was shown in Drosophila and in few other insect species by authors on systems, such as the nervous (Goodman and Bate, 1981; Abrams et al., 1993) and the muscular (Di Nardo et al., 1985), and in organs, such as the brain (Younossi-Hartenstein et al., 1993), the compound eyes (Wolff and Ready, 1991) and the ovary (Giorgi and Deri, 1976). Di Nardo et al. (1985) carried further the study of apoptosis during the development of the caudal region of the embryo in Drosophila melanogaster. Other authors (Ellis et al., 1981; Hengartner, 1994, 1996; White et al., 1994; White, 1995) have treated this subject in insects in general terms. On the other hand, there are no descriptions of the phenomenon of apoptosis in the dorsal organ of insects or in the systems or organs of cockroaches. The presence of some identical genes during programmed cell death in both vertebrate and invertebrate organisms suggests a stability of some apoptotic mechanisms during evolution. Furthermore, in addition to some stereotyped morphological and molecular features, uncommon aspects may be expressed, depending on the organism, cell type and means responsible for the death trigger (Palissot et al., 1996; Reipert et al., 1996). In the development of the dorsal organ during embryogenesis of Periplaneta americana, all the characteristic phases of apoptosis (chromatin and cytoplasm condensation, nuclear and cell cleavage) were found; nevertheless, some aspects may be considered unusual. Morphologically, both at the levels of light and electron microscopy, the initial phases of apoptosis were characterized by a condensation of chromatin in roundish bodies of different sizes, highly electron-dense and not always localized at the nuclear periphery. This is in contrast with the peripheral chromatin margination followed by the appearance of cap-shaped electron-dense areas generally described in the early stages of apoptosis (Wyllie et al., 1981; Falcieri et al., 1994a), and in our specimens only occasionally found. In spite of these morphological variations, the chromatin hypercondensation was accompanied by the activation of endogenous endonucleases and consequent genome cleavage. The chromatin fragmentation into oligonucleosomes is generally expressed during apoptosis, generat-

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FIGURE 7. Electrophoretic analysis (agarose gel electrophoresis) of nuclear DNAs obtained from the dorsal organ degenerating cell cluster. Lane M, DNA molecular weight marker VI (bands from 154 to 2176 bp); lane 1, DNA from the degenerating cells (the typical internucleosomal pattern of the apoptotic DNA cleavage is clearly visible).

ing a ‘ladder’ of DNA fragments detected by agarose gel electrophoresis (Wyllie, 1980; Compton, 1992). On the other hand, some authors have demonstrated that the DNA ladder does not always occur during programmed cell death in either vertebrate or invertebrate morphogenesis (Collins et al., 1992; Kerr et al., 1994; Tone` et al., 1994). Regarding the correlation between the appearance of

pyknotic chromatin, in the early phases of apoptosis, and internucleosomal DNA cleavage (Wyllie et al., 1984), recently it has been established that the expression of endogenous endonucleases is not fundamental in the triggering of apoptosis in some kinds of cells (Collins et al., 1992). The appearance of nuclear envelope protrusions surrounding microvesicular material during the phase of

APOPTOSIS IN SEROSA OF PERIPLANETA AMERICANA L. (BLATTARIA: BLATTIDAE)

chromatin condensation may be considered unusual. These structures may be correlated with functional changes of nuclear membrane components. Actually, during apoptosis, the perinuclear cisterna undergoes marked modifications (Falcieri et al., 1994b; Reipert et al., 1996) as a consequence of important changes in the nucleus-cytoplasm traffic (Wyllie, 1980). In spite of these changes, the karyological material never achieved complete pyknosis and a component, probably consisting of nuclear matrix and small amounts of residual DNA (Falcieri et al., 1994a) remained uncondensed. Consequently, during karyorrhexis, nuclear fragments, apparently chromatin-free, appeared. As apoptosis progressed, the cytoplasm underwent condensation and cleavage, forming membrane-bound bodies containing seemingly undamaged organelles. The apoptotic cell debris were ultimately recognized and internalized by large scavenger cells and subsequently degraded. These histiocytic cells, probably derived from vitellophages, appeared involved in the dorsal organ reabsorption through two ways. The primary scavenger activity was aimed at removing the potential toxic apoptotic cell fragments from the interstitial domain; the second involved the engulfment of cytoplasmic herniations arising from neighbouring cells, in which apoptotic degeneration was not yet triggered. The cellular herniation has recently been ascribed to the mechanisms that lead to the formation of cytoplasmic vacuoles during the endocytosis (Majno and Joris, 1996). This phagocytic activity, however, constitutes on the whole a helpful way of recycling certain molecules that are important as trophic support for embryo growth. The complex of these events in the controlled cell degeneration in embryogenesis of Periplaneta americana indicates that the mechanisms involved during apoptosis appear in part distinct from those described for other kinds of tissue and organism in invertebrate morphogenesis (Robertson and Thomson, 1982; Lints and Driscoll, 1996). This suggests that, depending on the biological situation, programmed cell death sequences may express departures from the stereotyped way. Apoptosis has been well characterized in several chordate species, but it has also been shown in a few invertebrate species (Milligan and Schwartz, 1996). This phenomenon appears therefore as an highly conserved evolutionary process with some fundamental steps, although the variations in some its morpho-functional features are still poorly described. REFERENCES Abbassy M. M., Helmy N., Osman M., Cope S. E. and Presley S. M. (1995) Embryogenesis of the sand fly Phlebotomus papatasi (Diptera: Psychodidae): cell cleavage, blastoderm formation, and gastrulation. Annals of the Entomological Society of America 88, 809–814. Abrams J. A., White K., Fessler L. I. and Steller H. (1993) Programmed cell death during Drosophila development. Development 117, 29–43. Bernardi R., Negri C., Donzelli M., Guano F., Torti M., Prosperi E. and Scovassi A. I. (1995) Activation of poly(ADP–

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Acknowledgements—The authors are grateful to Dr A. Mandarino, E. Clementi and A. Tronconi for their technical assistance. The work was supported by the Ministero dell’Universita` e della Ricerca Scientifica e Tecnologica, MURST.