Ecdysone: From Metabolism to Regulation of Gene Expression

1987.

INTRODUCTION: TEN YEARS OF ECDYSONE WORKSHOPS

RETROSPECT AND PERSPECTIVES

JULES A. HOFFMANN,     Endocrinologie et Immunologic des Insectes, UA 672, CNRS and Laboratoire de Biologie Générate, Université Louis Pasteur, Strasbourg, France

Publisher Summary

This chapter provides an overview of ecdysone workshops. The methods for the separation and analysis of ecdysteroids have undergone spectacular improvements, largely because of the introduction of high-performance liquid chromatography (HPLC) and the use of combined gas chromatography–mass spectrometry (GC–MS) of ¹H-NMR and ¹³C-NMR. Ecdysone and/or 20-hydroxyecdysone are inactivated primarily by the esterification or epimerization of the C-3 hydroxyl group or, to a lesser extent, by the esterification of the C-2 hydroxyl group on the A nucleus. A relatively wide-spread inactivation pathway is the hydroxylation of the C-26 methyl group followed by oxidation to an ecdysonoic acid. In higher insect orders, like the dipterans, ovarian ecdysteroids play a role in the control of vitellogenin synthesis, whereas in other orders, they play a role in the early events of embryogenesis. Vitellogenic ovaries synthesize ecdysteroids at a given stage of their development; in some species, this synthesis occurs only towards the end of vitellogenin uptake; in others, it occurs at almost the same time as concomitantly the onset of vitellogenin synthesis.

Ten years have elapsed since the first meeting in which ecdysone research played a significant part. In 1975, the CNRS had organized, under the auspices of Professors Durchon and Joly, an international conference of Invertebrate Hormones in Lille. Most laboratories engaged in ecdysone research throughout the world were represented at that conference at which 2 full days were devoted to ecdysone. This meeting undoubtedly had a marked impact on all those who attended, especially by creating new ties which many of us felt we should foster in the following years. This idea generated the ecdysone workshops, planned initially on a modest European basis, with the emphasis to be put on emerging or controversial points of research and with discussions given preference over formal papers. David Whitehead organized the first workshop, referred to as the second with respect to the Lille conference, at Imperial College in London in 1976. We were some 25 active participants at that time and the meeting lasted for 8 hr. In 1977, Peter Karlson and Jan Koolman invited us to the third workshop in a beautiful area of Bavaria. More than 70 scientists attended; and for the fourth workshop, organized in Strasbourg in 1979, we were more than 100 and a significant number of U.S., Japanese and Australian laboratories were represented. The workshops inevitably tended to become international conferences, but with a special family atmosphere which prevailed during the following meetings: Berne, superbly organized by Beatrice Lanzrein in 1981; and Szeged, which Peter Maroy masterminded in 1983.

In scientific research, 10 years is both a long and a short time. When reading through the proceedings of the Lille meeting, which appeared in early 1976, one is struck by the remarkable and often unexpected progress made in some aspects of ecdysone but one also feels dismayed to see that other subfields have remained almost stagnant.

As an introduction to this volume, it is suggested that, from the vantage point of Arthur’s seat in Edinburgh’s Salisbury Craigs, we look in two directions and ask two basic questions:

(1) What significant progress have we made in the 10 years between the Lille and Edinburgh meetings?

(2) What progress are we expecting to make in the next few years; which area of our research would we like to see develop and expand rapidly, and why? If I may express this latter question more romantically, I would ask: where in the field of ecdysone research are the golden apples of the Hesperides for which we are ready, like Hercules, to search for years and to shoulder the burden of Atlas? (not to speak of the necessity, before picking the apples, of killing the one hundred-headed dragon Ladon, which must be the equivalent in Greek mythology of present-day grant applications and administrative work).

To aid the reader who might be less familiar with the various aspects of ecdysone research, I should like to highlight four fields in which progress has undoubtedly been important during the last 10 years. These are: (1) Biosynthesis and metabolism of ecdysone and their regulation; (2) Mode of action of ecdysone at the cellular and molecular level; (3) Involvement of ecdysone in the regulation of reproduction and embryogenesis; (4) Phylogenetic aspects of ecdysone in protostomian metazoans. Before developing these four themes, we have to acknowledge the remarkable progress in technology which has permitted the advancement of ecdysone research since 1975. Radioimmunoassays of ecdysteroids, available in 1975 to a small number of laboratories, are now generally available and have allowed significant advances to be made. In particular, RIA now serves as a reliable tool for inhibition or stimulation assays of ecdysone-secreting glands (e.g. PTTH bioassay in prothoracic glands). The possibilities for the separation and analysis of ecdysteroids have undergone spectacular improvements, largely due to the introduction of high-performance liquid chromatography (HPLC) and the use of combined gas chromatography–mass spectrometry (GC–MS) of ¹H-NMR and ¹³C-NMR. The synthesis of high specific activity tritiated ecdysteroids (e.g. [³ H]ponasterone A) or ecdysone precursors has provided the biologist with powerful tools, as illustrated in this volume. The introduction of the most recent techniques of enzymology has made possible precise investigations of enzyme systems involved in ecdysone synthesis and metabolism (e.g. conversion of ecdysone to 20-hydroxyecdysone by the most thoroughly investigated enzyme in the ecdysteroid economy). Finally, the methodologies and concepts of molecular biology have provided ecdysone research with new dimensions which extend beyond the field of insect endocrinology.

Having paid a tribute to the advances in methodology, let us now look in some detail at the four domains of progress and the perspectives which will form the frame of this Introduction.

1 BIOSYNTHESIS, METABOLISM AND THEIR REGULATION

In 1975, after a period of several years of uncertainty, we had the firm demonstration in several insect spp. that the prothoracic glands are actually the predominant site of ecdysone biosynthesis during postembryonic insect development. In Crustaceans, the Y-organs were reported at the Lille meeting to be an ecdysone-producing gland. We have since learned that in reproductively-competent females the ovaries produce ecdysone and the exact site of biosynthesis has been shown, at least in two insect species, to be the follicle cell epithelium. As opposed to these established sites of ecdysone biosynthesis, we have evidence that, under certain circumstances (e.g. pupal development of Coleopterans; long-term cultures of epidermis in vitro; and some prothoracectomized or neck-ligatured larvae) other tissues must have some capacity, either to release stored ecdysteroids, or possibly to synthesize these molecules de novo. We know also that during embryogenesis many insects can increase their titre of ecdysone by one or two orders of magnitude in the absence of any differentiated established site of ecdysone biosynthesis. The tissues which are involved in these processes, and the biochemical pathways by which they occur, are among the challenging subjects of present research.

Although we have known for a long time that cholesterol (originating either from the diet or from the conversion of a dietary phytosterol) is the common biosynthetic precursor of ecdysone, our progress on the biogenetic sequences leading from cholesterol to ecdysone has not been as complete over the last 10 years as might have been expected. Figure 1 summarizes our present understanding which is divided, following an idea of Dennis Horn expressed at the IVth Ecdysone workshop, into a black box and a bright box. At present we are still uncertain about the pathways which transform cholesterol to the relatively mature deoxyecdysteroid, the 5β-ketodiol (or 2,22,25-trideoxyecdysone). In other words, such essential events as the introductions of the 6-keto group, of the Δ7 double bond, of the proton at C-5 (and concomitant A/B cis ring junction) and the hydroxylation at C-14α remain obscure to us.

Fig. 1 Pathways of biosynthesis of ecdysteroids. Most insect species synthesize ecdysone and 20-hydroxyecdysone. Hemipterans and Hymenopterans produce makisterone A. Crustaceans synthesize ponasterone A in addition to ecdysone and 20-hydroxyecdysone. Some insect ovaries produce 2-deoxyecdysone predominantly. For black box, see text.

In contrast to this, we have reasonable information on the probable sequence of events of hydroxylations on the side-chain (C-25, followed by C-22) and we know that hydroxylation at C-2 is the last step of ecdysone biosynthesis, at least in the models so far investigated. Studies of the enzymes involved in ecdysone biosynthesis are in their infancy; this volume includes a contribution which shows that the C-2 hydroxylation system is mitochondrial and exhibits some (but not all) of the characteristics of a cytochrome P-450-dependent monooxygenase. Interestingly recent evidence is in favour of a microsomal localization for the side chain hydroxylation systems (C-22, C-25).

Undoubtedly the final establishment of the complete biosynthetic pathways of ecdysone and the full biochemical characterization of the enzyme systems involved, their cellular and subcellular localization, and their regulation are among the golden apples of ecdysone research.

Compared to the relative uncertainty surrounding ecdysone biosynthesis, we have experienced a marked progress in recent years in our knowledge of ecdysone metabolism. One of the obvious reasons for this discrepancy in our understanding relates to technology, as explained above: while [³H]ecdysone of high specific activity has been available for more than a decade, this was not the case until recently for putative precursors. Figure 2 summarizes our current view of ecdysone metabolism in insects. The reader will find several contributions concerning comparative aspects in this volume. We will tentatively separate the metabolic reactions of Fig. 2 into three groups:

Fig. 2 Main routes of ecdysone metabolism. For explanations, see text.

Hydroxylation of ecdysone at C-20 to 20-hydroxy-ecdysone

At the Lille meeting it was proposed that this reaction should be considered as an activation mechanism, converting the poorly active prohormone ecdysone to the 10–100 times more potent 20-hydroxyecdysone. Relative activities in this context refer mainly to assays using Dipteran tissues or cell lines in vitro. This interpretation has been, and still is, challenged in view of results obtained with other biological models where ecdysone and 20-hydroxyecdysone apparently exert different and complementary actions. We may leave open the possibility at this stage that more primitive insects use both ecdysone and 20-hydroxyecdysone as hormonal messengers, but during evolution ecdysone lost its proper hormonal role and is merely a prohormone in Dipterans. But, obviously, more experimentation is needed in this area, and an ideal tool would be a selective inhibitor of the 20-monooxygenase, a present goal of several research groups. This enzyme is by far the best known of all enzymes involved in ecdysteroid metabolism. It is localized in many peripheral tissues, like the fat body, Malpighian tubules, gut etc., but is absent from prothoracic glands (and, in contrast, it is present in follicle cells, another established site of ecdysone biosynthesis). This volume contains several contributions on ecdysone-20-monooxygenase. Suffice it here to say that there is evidence that, dependent on the insect spp. and tissue, there exist both a mitochondrial and a microsomal 20-monooxygenase. They have the characteristic properties of cytochrome P-450-dependent monooxygenases. The C-20 hydroxylation of ecdysone undergoes sharp fluctuations during development and it has been suggested that these might be substrate-induced, that is, depend on the rise of the concentration of ecdysone which ultimately is regulated by PTTH, the prothoracicotropic neurohormone (see below). Again, additional experimental work is needed before any final conclusions can be drawn.

Inactivation mechanisms

Ecdysone and/or 20-hydroxyecdysone are inactivated primarily by esterification (acetylation, phosphorylation, …) or epimerization (oxidation followed by isomeric reduction) of the C-3 hydroxyl group or, to a lesser extent, by esterification of the C-2 hydroxyl group on the A nucleus. A relatively wide-spread inactivation pathway, which was only recently discovered, is the hydroxylation of the C-26 methyl group followed by oxidation to an ecdysonoic acid. An increasing number of results from a variety of arthropods and non-arthropods, which the reader will find in this volume, point to the possibility of esterification of the C-22 hydroxyl group by various fatty acids. Such esters are probably inactivation products in many cases, but we have to leave open the possibility that in some instances they might be reused in vivo after conversion by esterase activity.

Storage mechanisms

Over the last few years it has become apparent that a large proportion (up to 98% in some spp.) of the ovarian ecdysteroids are esterified to phosphate conjugates at the C-22 hydroxyl group. More complex conjugates bearing nucleotides on the phosphates are occasionally, but not regularly, found together with the 22-phosphates. These ovarian conjugates are present in newly-laid eggs and, as we shall see below, are gradually metabolized during embryogenesis. Based on this finding (restricted to a very limited number of insect spp. and to experiments indicating that the egg contains enzymes capable of hydrolyzing 22-phosphate conjugates) it is a common belief that these molecules represent storage forms. We will come back to this problem in the third section. In ticks, eggs contain 22-acyl esters but no 22-phosphates. Whether or not these can also be considered as storage forms is still a matter of debate.

Finally, to close this section on ecdysone metabolism, we should stress that our information on the tissue localization of the enzymes involved in ecdysteroid metabolism has noticeably increased in recent years. Figure 3 summarizes the most relevant data available from insects.

Fig. 3 Tissue localization of major biosynthetic and metabolic reactions of ecdysteroids in insects. Note that in many insects prothoracic glands are no longer functional when the ovaries start synthesizing ecdysteroids.

The regulation of ecdysone biosynthesis is one fascinating field which is experiencing rapid progress. The isolation and elucidation of the structure of prothoracicotropic neurohormone (PTTH) is well advanced in lepidopteran larvae and pupae. An unexpected result of these studies of Bombyx and Manduca was the finding that in each spp. two PTTHs are present: a small PTTH (4 kD in Bombyx;7kD in Manduca) and a large PTTH (22 kD in Bombyx; 29 kD in Manduca). This raises the intriguing possibility that these two PTTHs might have different roles in the control of post-embryonic development. Whether the two PTTHs are processed from a common precursor or are products of different genes expressed in different neurosecretory cells is another exciting question presently under investigation.

Among the interesting facets of these recent results is the observation that the amino-acid sequence of 19 amino-acid residues at the amino terminus of the small (4 kD) PTTH of Bombyx show significant homology with insulin and insulin-like growth factors. The authors of this finding suggest that a common ancestral peptide might have diverged in the course of evolution into peptides with different functions in insects and vertebrates.

The purification of PTTH in lepidopterans now makes possible studies on the mode of action of the neurohormone, and this volume includes a contribution which suggests that calcium plays a primary role in mediating a PTTH-stimulated synthesis of cyclic AMP, which in turn stimulates ecdysone synthesis.

It would certainly be an oversimplified view to regard PTTH as the sole regulator of ecdysone biosynthesis. Reports in this volume indicate a role for juvenile hormone which exerts, at least in Manduca, an indirect tropic effect on the prothoracic glands, probably mediated by a 30 kD stimulatory protein in the haemolymph which could be a precursor steroid carrier. Also the intrinsic developmental competence of prothoracic glands to respond to the regulatory effectors appears to be a key factor in the regulation on ecdysone synthesis. The reader will find in this book a model which explains how primary and secondary regulatory effectors, hormone interactions, and gland competence may all be integrated to regulate the ecdysteroid and juvenile hormone titres during larval–pupal development in Manduca.

In contrast to our rapidly expanding information of PTTH (restricted so far to the Lepidoptera), we have very few data on adult ecdysiotropins. There is experimental evidence for the existence of a neuroendocrine regulation of ecdysteroid synthesis in ovaries of Aedes and Locusta, but the isolation and subsequent identification of these molecules is still in its infancy. It may be significant that extracts of brain-Corpora cardiaca complexes from vitellogenic females of Locusta have the capacity to stimulate the synthesis of ecdysone in prothoracic glands in a dose-dependent manner, which suggests structural similarities between the larval (PTTH) and adult ecdysiotropins in this species.

We suspect that the embryo has developed a sophisticated ability to regulate its ecdysone titres. We will come back to this problem in the third section of this Introduction.

Obviously, the comparative analysis of the molecules which control steroid biosynthesis and release in embryos, larvae, pupae and adults, in the various insect orders, and in other Arthropods, is one of the most fascinating areas of ecdysone research, one of the golden apples. The establishment of reliable and rapid assays for these molecules will eventually enable us to determine which external and internal factors turn on the whole cascade of events which lead from neurohormone synthesis and release to the expression of an ecdysone-induced gene. A golden apple, indeed.

2 MODE OF ACTION OF ECDYSTEROIDS: MOLECULAR ASPECTS

At the Lille meeting 10 years ago, we heard a communication concerning ecdysone-binding in ecdysone-sensitive Drosophila Kc cells; this binding was apparently not observed in insensitive cells. Whatever the shortcomings of these first experiments, a variety of subsequent studies established beyond any doubt, between the first and fourth Ecdysone workshops, that the target tissues of ecdysone contain proteins which bind ecdysone or related molecules. The binding was shown to have what are now seen as the classical characteristics of steroid hormone receptors, namely: high affinity for the hormone, and a limited number of binding sites per target cell. In addition, the binding proteins exhibited a differential affinity for ecdysone analogs which paralleled the different activities of these analogs in eliciting known biological effects. This sort of result, based on the use of radioactive ligands (ponasterone A proved a most valuable tool in this respect) was reported for Dipteran tissues and cells (imaginal discs, fat body, Kc cells) and for various Crustaceans’ tissues (gut, epidermis). It was logical to infer that these binding proteins corresponded to ecdysteroid receptors, and in consequence efforts have been directed towards the isolation of the ecdysteroid receptors. This undertaking however is still hampered by a series of methodological difficulties which have not yet been fully overcome.

Let us now turn our attention to the mode of action of ecdysone at the genome level. Ten years ago the discussions at Lille centred on the Karlson model, based on a series of pioneering studies with Chironomus and Drosophila which showed that in polytene chromosomes certain stage-dependent puffs could be induced by the injection of ecdysone. In this model it was assumed that the hormone, or the hormone-receptor complex, bound to chromatin close to the coding DNA of ecdysone-inducible genes, causing a rapid unwinding of large stretches of chromatin (puffs) and the initiation of mRNA synthesis. Puffs derive from chromosome bands and normally involve a length of 10–100 kb of DNA, which meant that, if one band corresponded to one gene, then the eucaryotic gene would be considerably larger than the procaryotic gene, which has an average of 1–3 kb of coding DNA. This difference might result from differences in the extent of regulatory sequences.

Since the Lille meeting our understanding has increased considerably, thanks to a series of biological systems all derived from Drosophila. These include the induction and repression by ecdysone of synthesis of glue proteins in salivary glands and of several proteins in fat body, control by ecdysone of evagination and cuticulogenesis in mass-isolated discs and cell alterations, enzymatic changes and protein synthesis provoked by ecdysone in established Kc cell lines. In the past few years it has been shown, both by genetic analysis and by molecular cloning techniques in several of these systems, that the transcribed sequences of ecdysone-induced genes in several cases are not significantly longer than the coding sequences of procaryotic genes. It has also been confirmed by molecular techniques that ecdysone-controlled genes in Drosophila contain exon and intron sequences, and so resemble many other eucaryotic genes. The finding that a single gene could apparently give rise to a number of transcripts of different sizes suggested the hitherto unsuspected possibility that differential splicing of primary gene transcripts may occur and that control of gene expression by ecdysone may occur at this post-transcriptional level. To date, however, we have no experimental evidence that ecdysone does play a role in the control of gene splicing.

In the last 2 or 3 years considerable effort has been invested in unraveling the composition of the regulatory sequences of ecdysone-inducible genes in Drosophila. These efforts have immensely benefited from the introduction of P-element transformation vectors. The rationale of this experimental system is schematized in Fig. 4. P elements are factors implicated in the phenomenon of hybrid dysgenesis. The complete P element has a coding sequence of 2.9 kb and a 31 base pair inverted repeat at each end. In 1982 Rubin and Spradling constructed a vector by inserting such a P element into a plasmid. The P elements exise precisely from the plasmid when injected into germ cells of embryos (thanks to a transposase coded for by a P factor) and a certain percentage become integrated into the germ-line chromosomes. The P element has unique restriction sites (as for example the Xho I site shown in the P element of Fig. 4) which can be used to introduce an ecdysone-induced structural gene into the P element construct. This gene, once integrated into the host genome, will be expressed in the progeny of transformed embryos (a few percent of those injected), provided that sufficient gene flanking sequences were introduced into the P element. By manipulating the gene flanking sequences in vitro with molecular biological techniques prior to insertion into the P element and subsequently testing the functional capacity of the modified sequences, we can hope to establish the length and composition of the regulatory sequences of ecdysone-inducible genes. Results obtained with glue gene studies in salivary glands and the P1 gene in fat body indicate that, in these two model systems, the regulatory sequences are considerably longer than bacterial gene promoters (average 0.1 kb) and possibly involved as much as 1.5 kb of the 5′ flanking region.

Fig. 4 P-element transformation vectors. On the left, a P element has been introduced into a plasmid vector. The Xho I restriction site in the P element has served to introduce on the right of the figure an ecdysone-inducible gene (structural gene) with 5′ and 3′ flanking sequences. The introduction of a marker gene (in this example the rosy gene, ry+) helps to screen for the positive integration of the transposons into the germ line cells and their expression in the progeny. For other details, see text.

The P element transformation system makes the study of the ecdysone-inducible genes currently one of the most powerful eukaryotic systems for the establishment of regulatory sequences, and once receptors have been purified it should lead to studies of binding of receptors to these sequences. The reader will find these problems discussed in detail in this volume.

It became apparent at the workshop that, as opposed to the relatively simple ecdysone-inducible genes of salivary gland glue proteins or the P1 larval serum proteins of fat body, which each have a single transcript, other ecdysone-inducible genes show a complex pattern of transcription and stretch over regions of 50 kb or more. The EIP 28/29 gene (coding for ecdysone-inducible polypeptides 28 and 29 kD) investigated in established Kc cell lines of Drosophila, for instance, is embedded in a nest of overlapping tissue-specific transcription units which are not regulated by ecdysone. The controlling elements for the EIP 28/29 gene are included on a fragment containing the transcription unit and 1–2 kb of flanking DNA at each end. Similar complexities are apparent in the transcripts from the region of the ecdysone-inducible puffs 2B, 74 EF and 75B. These salivary gland early puff transcripts are extremely long, of the same order of magnitude as the puffs themselves. The region containing the EIP 40 gene (ecdysone-inducible polypeptide of 40 kD) is transcribed in both directions in Kc cells and both of the transcipts are ecdysone-inducible.

The VIIth Ecdysone workshop has clearly shown the increasing impact of molecular biology in the field of ecdysone research. We can now reasonably hope that the analysis of the regulatory processes of ecdysone-inducible proteins by molecular-biological techniques will lead us eventually to understand the logic of stage- and cell-specific gene expression in the course of insect development, really one of the golden apples, interest in which will extend far beyond the area of insect biology.

3 INVOLVEMENT OF ECDYSTEROIDS IN THE CONTROL OF REPRODUCTION AND EMBRYOGENESIS

At the time of the Lille meeting we heard or knew that, in at least 3 insect spp, ovaries of reproductively competent females produced and/or accumulated ecdysteroids. This has been confirmed over the last 10 years in a large variety of insect and crustacean spp., and we tend to believe to-day that it is probably a general feature among arthropods. The initial reports on ovarian ecdysteroids met with some scepticism and, although progress in this field has been considerable, no clearcut picture has evolved, possibly because there might be profound differences among the various orders with respect to the destiny and role of ovarian ecdysteroids.

Let us first summarize the evidence: vitellogenic ovaries synthesize ecdysteroids at a given stage of their development; in some species (orthopterans) this synthesis occurs only towards the end of vitellogenin uptake; in others, as in some dipteran spp., it occurs at almost the same time as concomitantly the onset of vitellogenin synthesis. As mentioned earlier, in at least two species it has been demonstrated experimentally that the ovarian ecdysteroids are synthesized by the follicle cell epithelium. Although we assume that this is true for the other species as well, it remains to be proven. Obviously these ecdysteroids can have two destinies: either they are released into the blood of the female or they accumulate inside the oocytes and are present in the newly-laid eggs. The available data indicate that this double destiny is found in most species. However, the ratio of the amounts released into the blood to those retained in the oocytes seem to vary enormously between insect orders: in locusts for example, only one out of 50 molecules produced by the ovary is released into the blood of the female, the others accumulating inside the oocytes and consequently being present in the newly-laid eggs; while in Galleria, this ratio is 1:1. The situation is further complicated by the fact that in most species the majority of ovarian ecdysteroids are esterified and have often escaped detection or have been seriously underestimated in quantitative studies.

A current view, extremely simplified for the reader, is that in higher insect orders, like the dipterans, ovarian ecdysteroids possibly play a role in the control of vitellogenin synthesis, whereas in other orders they are destined to play a role in the early events of embryogenesis. Some additional roles have been proposed in a variety of species, e.g. separation of incipient follicles from the germarium, and control of release of ovulation hormone in mosquitoes.

As regards the possible involvement of ecdysteroids in the control of vitellogenesis in dipterans, there is indeed ample experimental evidence that in this order ecdysteroids can stimulate the synthesis of vitellogenin mRNA, both in females and in males. Correlative studies of hormone titres and of vitellogenin synthesis in vivo in dipteran females are compatible with the idea that circulating ecdysteroids can stimulate the fat body to synthesize increased amounts of vitellogenin. It is possible that the fat body has to be primed by juvenile hormones to become responsive to ecdysteroids in this regard.

The assumption that ovarian ecdysteroids play a role in the early events of embryogenesis is based on a series of investigations, performed mainly on orthopterans, which can be summarized as follows: remarkably high concentrations (∼ 10−4 M) of ecdysteroid conjugates of ovarian origin are present in newly-laid eggs. As embryogenesis proceeds these molecules are transformed into other ecdysteroids identical to the inactivation forms described in the first section. The presence of enzymes responsible for these transformations has been monitored in locust eggs by the use of labelled compounds. One among many reactions occuring during locust embryogenesis can serve to illustrate this: newly-laid eggs contain large concentrations of a 22-phosphate of 2-deoxyecdysone, but no 3-phosphate of the 3-epimer of 2-deoxyecdysone. The latter compound is almost certainly an inactivation product. As embryogenesis proceeds, the 22-phosphate of 2-deoxyecdysone is replaced by the esterified 3-epimer of 2-deoxyecdysone. The replacement is not gradual, but particularly intense at certain times for reasons still poorly understood. The obvious conclusion from this sort of observation is that the maternal conjugate is hydrolysed to the free ecdysteroid needed to trigger some specific events before being in turn metabolized to an inactivation product. Now indeed, when assaying the titres of free ecdysone during embryogenesis in various insect species, it is apparent that there are a certain number of peaks of concentration of ecdysone and/or 20-hydroxyecdysone. Interestingly, in all cases investigated, these peaks were found to be coincident with cuticle depositions, either by the serosa or by the embryonic epidermis. In fine, the sum of these observations has led to the following working hypothesis: via a large supply of ecdysteroid conjugates the mother controls ecdysone-triggered events of embryogenesis which occur prior to the (still unknown) stage when the embryo has acquired its own capacity to biosynthesize ecdysone de novo; one of these ecdysone-triggered events is cuticulogenesis. Such a transfer of hormonal messengers from the mother to the embryo would be novel amongst oviparous organisms and it has stimulated considerable interest. For the developmental biologists it raises several unexpected questions, among which the major one concerns the regulation of the hydrolysis of the conjugates in the early phases of egg-development. Also, the role of the transferred maternal ecdysteroids is certainly not restricted to the control of embryonic cuticulogenesis. The decade since the Lille meeting has increased our information on the phenomenology of ovarian ecdysteroids enormously. We must hope that the next years will be informative as regards the roles and mode of action of these molecules. Approaching these problems with molecular biological techniques offers us another of the golden apples for which we are looking.

4 PHYLOGENETIC ASPECTS: ECDYSTEROIDS IN NON-ARTHROPOD METAZOANS

At the time of the Lille meeting we already knew that ecdysteroids were present in a variety of plants: pteridophyta, gymnosperms, and angiosperms. The physiological relevance for the plants of these so-called phytoecdysteroids (many of which are strictly identical to the zooecdysteroids) is a matter of debate. A high concentration of such molecules probably protects some roots or barks against predatory insects, although this is not firmly established.

There were occasional reports in the seventies on the presence in some worms of molecules claimed to be ecdysteroids, but as the methodologies which were used were not reliable, those reports raised only limited interest. Since the two most recent ecdysone workshops however, considerable efforts have been made to analyse the phylogenetic aspects of ecdysone research, and it has now been established using adequate techniques that many protostomians contain ecdysteroids. The list is given in Fig. 5, and indeed one wonders if the presence of ecdysteroids may not be universal among invertebrates.

Fig. 5 The presence of ecdysteroids among the various metazoan phyla: positive identifications with physico-chemical methods are reported from the groups which are boxed.

The molecules which were identified in non-arthropods are similar to those with which we are familiar in different insects: ecdysone, 2-deoxy-ecdysone, 20-hydroxyecdysone and 20, 26-dihydroxyecdysone. This list will certainly become longer at the next workshop. As in arthropods, these ecdysteroids are found both under free or esterified form, the esterifying moieties being either polar or non-polar. The titres recorded for ecdysteroids in non-arthropods are generally lower than those found in arthropods. Titre fluctuations have recently been recorded in relation to integumental events, to reproductive cycles and to embryonic metamorphosis. Obviously this has led to speculations that in non-arthropods ecdysteroids might play roles similar to those which we know them to exert in arthropods. This is an exciting speculation, but we must be aware that today it is merely a speculation. What we badly need in the near future are experimental data on the origin and the mode of action of ecdysteroids in non-arthropods before we can conclude that the various phyla listed in Fig. 5 actually use these molecules as hormonal messengers, as do arthropods. This is more likely to be shown to be the case in worms rather than in molluscs.

Although this area of phylogenetic research is in itself rewarding for the biologist, considerable interest arose when it was discovered that human patients infested by trematodes, cestodes and nematodes contained ecdysteroids in their blood and urine. The potential of this finding for diagnosis and possibly even for interfering with the parasite’s development or reproduction has caused some excitement in our scientific community. It is probable that radio-immunoassaying ecdysone in sera or urine of humans will contribute to diagnosis of helminth infection. As it is still a very open question whether or not these parasites use ecdysteroids for the control of their reproduction and for development, it is premature to be confident that these data open a novel way of fighting parasitic infection. But it is certainly worthwhile to explore this.

In 1934, in his study on the "Physiology of ecdysis in the Hemipteran Rhodnius prolixus," V. B. Wigglesworth established the existence of a hormone controlling moulting and metamorphosis. One year later, G. Fraenkel published a study of the hormone causing pupation in the blowfly Calliphora erythrocephala. We know from P. Karlson’s autobiography that late in 1943 he started the work on the isolation of the moulting hormone of insects, using Fraenkel’s studies as the basis of the hormone bioassay. Some ten years later, in 1954, Butenandt and Karlson had succeeded in isolating crystalline ecdysone and by 1965 the structure of the hormone had been fully elucidated, only 20 years ago! It was not until 1975 that a major meeting was able to gather together 30 scientists involved in ecdysone research all over the world. Looking at the present volume, the reader will sense that the progress has been almost exponential in the 50 years since Wigglesworth discovery. And yet the number of unsolved questions remains impressive. We will concur modestly with the ancient philosopher: while we have no final answers, at least we have progressed in learning how to ask the right questions!

Acknowledgements

I am indebted to Dr Geoffrey Richards, Dr Charles Hetru, Dr Maurice Charlet and Dr Marie Lagueux for stimulating discussions and to Mrs Colette Boiler for the preparation of the manuscript.

Addendum—This article is meant as a general introduction to this volume and does not include references. Recent review articles can be found in: Biosynthesis, Metabolism and Mode of Action of Invertebrate Hormones (Edited by Hoffmann J. A. and Porchet M.). Springer Verlag, Heidelberg (1984) and Invertebrate Endocrinology, Vol. I, Endocrinology of Insects (Edited by Downer R. and Laufer H.). Alan Liss, New York (1983).

ECDYSTEROID METABOLISM

A COMPARATIVE STUDY

R. LAFONT¹, P. BEYDON¹, C. BLAIS¹, M. GARCIA¹, F. LACHAISE¹, F. RIERA¹, G. SOMME¹ and J.P. GIRAULT²,     ¹Ecole Normale Supérieure, CNRS U.A. 686, 46 rue d’Ulm, 75230 Paris Cedex 05; ²Université René Descartes, CNRS U.A. 400, 45 rue des Saints Péres, 75270 Paris Cedex 06, France

Abstract

The metabolism of ecdysone and 20-hydroxyecdysone has been investigated in various animal species, including representatives of arthropods, molluscs, annelids and mammals. Some of the major metabolites have been isolated and characterized by mass spectrometry and two-dimensional proton NMR.It appears that metabolic pathways differ strongly between species and that each class has evolved specific reactions.

Key Word Index:

Ecdysteroids

metabolism

insects

invertebrates

INTRODUCTION

Ecdysteroids are common amongst invertebrates and plants (Karlson, 1983) and in arthropods it is well-established that they function as hormones controlling development and reproduction. In other groups of invertebrates, however, the evidence for their presence is not accompanied by evidence for their endogenous origin or their hormonal function, and we cannot presently deny that in many cases they may derive from the animal’s food.

Besides this important question regarding the physiological significance of ecdysteroid presence in animals, we may question whether all invertebrates contain the same compounds and have evolved the same metabolic pathways for these steroids. In addition, we may ask whether similar or equivalent reactions can take place in vertebrates. This last point may be of practical importance in the case of mammals, including man. It appears that in mammals infested with helminths, infestation could be detected by analysing ecdysteroids in their blood or urine (Nirde et al., 1984; Koolman et al., 1984). However, if ecdysteroid metabolism in mammals is rapid, it would be advisable to search instead for some more abundant metabolite(s). Moreover, as there is increasing evidence for pharmacological effects of ecdysteroids on mammals (Catalan et al., 1980, 1984), it could be of interest to determine whether these molecules can be converted into compounds somewhat similar to vertebrate steroids.

Considering the high variation in the chemical structure of ecdysteroids isolated from plants, where more than 60 different molecular species have been characterized (Bergamasco and Horn, 1983), we may question whether the same variety also occurs among invertebrates.

In order to answer this question we have started an extensive analysis of ecdysone and 20-hydroxyecdysone metabolism among various species representative of different animal groups: arthropods (insects, crustaceans, arachnids), molluscs (gastropods), annelids (oligochaetes) and vertebrates (mammals). The reactions which take place in insects have been reviewed recently (Lafont and Koolman, 1984). Several types of reactions have been considered in this study: the presence of an hydroxylation at the C-20 position; the presence of C-26 hydroxylation, followed by ecdysonoic acids formation; the presence of other hydroxylation reactions, and their positions; the possible oxidation or reduction of the molecule; the formation of 3-epimers; the formation of hydrolysable conjugates; and the cleavage of the side-chain.

We wish to report here especially on ecdysteroid metabolism in some non-arthropod species. The present study has involved the isolation of metabolites formed after injection of exogenous ecdysteroids together with a ³H-labelled marker. The major metabolites have been characterized by mass spectrometry and two-dimensional proton NMR.

MATERIALS AND METHODS

Biological materials

The various species used were purchased by various suppliers (mice, Mus musculus; earthworms, Eisenia foetida); collected in the field (terrestrial snails, e.g. Cepaea nemoralis, Helix aspersa; earthworms, Allolobophora sp., crabs, Carcinus maenas; scorpions, Androctonus australis, Buthus occitanus), or raised in the laboratory (insects, Pieris brassicae, Locusta migratoria, Calliphora erythrocephala, Drosophila melanogaster, Carausius morosus).

Chemicals

Unlabelled ecdysone and 20-OH-ecdysone were purchased from Simes (Milan, Italy). Reference 26-hydroxy-ecdysone was isolated from Manduca sexta eggs (a gift from Dr Bernard Mauchamp). Ecdysonoic acids were isolated from Pieris brassicae pupae (Lafont et al., 1983). Reference 3-dehydroecdysteroids were prepared by incubation in vitro of the corresponding 3β-hydroxy-compounds with cytosolic preparations from Pieris pupae (Blais and Lafont, 1984). Reference 3-epimers of ecdysone and 20-OH-ecdysone were prepared by incubation of 3β-compounds with Pieris larval gut homogenates.

Labelled ecdysone and 20-OH-ecdysone were prepared in vitro by incubation of [23,24-³H] 2-deoxyecdysone (a gift from Dr Charles Hetru) with adult male locust Malpighian tubules (Modde et al., 1984).

Hormones were injected in isotonic NaCl solution (containing 10% isopropanol in the case of mice, where large amounts of ecdysteroids were injected). Subsequent analysis of metabolites was performed using whole animals or faeces (invertebrates), or urine, faeces and various organs (mice).

Analysis and isolation of metabolites

Ecdysone metabolites were extracted according to Lafont et al. (1982). They were then analysed by normal or reverse phase HPLC (Lafont et al., 1981). Radioactivity in the eluent was monitored using a FLO-ONE model IC on-line detector (Radiomatic Instruments, Tampa, U.S.A.).

Large-scale isolation of metabolites involved solvent partition, SEP-PAKRC-18 purification (Waters Inc.) preparative reverse phase HPLC and, finally, normal phase HPLC (see e.g. Garcia et al., 1985). Purified metabolites were then analysed by mass spectrometry and proton NMR (see e.g. Diehl et al., 1985; Garcia et al, 1985).

RESULTS

The results of our experiments are reported in Table 1. It must be noted that for several groups they were obtained from a few species only, and that any generalization would be premature.

Table 1

Observed metabolic conversions of ecdysteroids by various