CANCER RESEARCH...cuticles in Rhodnius prolixus, and Fraenkel (43) reported similar findings for Calliphora. Weyer (115) demon-*Aided by a grant from the Department of Health, Education,

CANCER RESEARCH

VOLUME24 MAY 1964 NUMBER4

The Significance of Invertebrate Hormones in Relationto Differentiation*

WALTERJ. BÃœROBOTE(Department of Surgery and Laboratory of Clinical Biology, University of Utah College of Medicine, Salt Lake City, Utah)

The manner in which specialized form and function areexchanged in a predictable pattern for the pluripotentialityof the zygote poses one of the most refractory riddles inbiology. A few generalizations concerning the steps inconverting the polymorphic to the differentiated organismhave emerged, but the total concept remains inadequate.On the assumption that common mechanisms, withdifferences only hi the details, may operate in all biological systems of differentiation, it may be profitable toexamine information contributed by invertebrate systemsof metamorphosis to the general knowledge.

The endocrine systems that exist hi invertebratesconstitute a fascinating chapter hi the history of science.Some of them are exotic but not germane to the topicunder discussion. For example, pheromones (3, 27, 28,32, 66, 68), such as the sex attractants (26, 30, 31), behavein many respects like hormones but exert their effectat a distance to produce a lepidopteran romantic telemetrythat is the envy of purveyors of perfume from Paris toCairo. Others are intimately related to the process ofdifferentiation.

INVERTEBRATE HORMONES

In invertebrates, the process of growth necessitatesmoulting and the periodic renewal of the exoskeleton.Attention will be directed toward three groups of hormones and the tissues that produce them, since they aredirectly concerned with the regulation of events takingplace during moulting and metamorphosis. The orderlyinitiation and succession of the usual pupal, larval, andimaginai stages in the process depend on neurosecretionfrom the brain, juvenile hormone (neotenin) from thepaired corpora aliata, and ecdysones from the prothoracicglands.

Brain hormone.â€”Early in the twentieth century theconviction prevailed that insects do not secrete hormones.However, KopeÃ¶was able to show by 1917 that pupationis initiated by a hormone (86) secreted in the brain.Wigglesworth (117) confirmed the presence of a hormonein the pars intercerebralis of the protocerebrum thatinduced each cycle of growth and formation of successivecuticles in Rhodnius prolixus, and Fraenkel (43) reportedsimilar findings for Calliphora. Weyer (115) demon-

* Aided by a grant from the Department of Health, Education,and Welfare, U. S. Public Health Service.

521

strated secretory activity in the brain of Apis nielli/eraat about the same tune. Others (70, 103, 114) haveconfirmed and extended these observations. Fukuda(44) demonstrated that the moulting hormone hi thesilkworm was produced by the prothoracic glands. Therelationship between this discovery and the known effectsof neurosecretion on development was worked out byWilliams (120) in Platysamia. He found that the pro-thoracic glands secreted hormone hi response to stimulation by secretion from the brain.

Neurosecretory droplets have been noted hi the corporacardiaca at the termination of axons from the brain byboth HanstrÃ¶m (57) and Scharrer (101). It is usuallyassumed that the pathway for release is through thecorpora cardiaca. However, noting that two pathwaysfor neurosecretion (70) are visible from the onset of fourthand fifth larval stage of the silkworm, one through theaxon of the A-cell and the other to the open circulationvia the cortex, Kobayashi suggested that the prothoraco-tropic hormone concerned with imaginai differentiation isprobably released through the latter pathway (71). Ichi-kawa and Nishiitsutsuji-Uwo found that corpora aliataalso store neurosecretory material transported via thenerves connecting them with the brain (60), and Kobayashi et al. (72, 77, 84) have reported imaginai differentiationof Dauer-pupae in the presence of corpora aliata. Withhis collaborators at the Sericultural Institute in Tokyo(69, 78, 79), Kobayashi was able to isolate (from thousandsof brains dissected from silkworms) cholesterol, hi addition to a protein fraction, as an active material producingthis effect. This action of cholesterol has been confirmedby Schneiderman and co-workers. However, hormonefrom the brain may also have a direct effect on tissues(61, 89) hi influencing the course of metamorphosis. Forexample, Kobayashi and Burdette (73) found that pupation of Calliphora was produced when brain hormonewas injected with hormone from prothoracic glands inconcentrations of the latter that produced no effect. Contamination of the former with the latter in the process ofextraction does not seem probable, since high concentrations of brain hormone did not produce a similar positivebioassay.

Both Bergman (12) and Karlson (65) report isolationof cholesterol in relatively large amounts from the silkmoth, and the latter questions whether cholesterol represents the active principle secreted bv the brain. The

This One

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522 Cancer Research Vol. 24, May 1964

likelihood that cholesterol is a precursor of ecdysone hasbeen enhanced by the observation of Church (35) andWigglesworth (118) that continued neurosecretion isnecessary for the orderly progression of metamorphosis.By using labeled cholesterol, Karlson and Hoffmeister(67) have demonstrated recently that this is the case. Itshould be noted, however, that Kobayashi suggests thatcholesterol from the brain may be different from thatfound elsewhere in the insect, since the latter is presentduring diapause. A number of active extracts of brainhormone have been obtained by investigators other thanthe group at the Sericultural Institute. The extracts ofGilbert and Schneiderman (53) as well as Kobayashi et al.(69, 78-80) are lipide-soluble, whereas Gersch (46, 47)and Ichikawa (59) and co-workers report activity infractions soluble in water. Thus the hormonal chemistryof neurosecretion in insects remains partially unresolved.Activity resembling that of the brain hormone on Dauer-pupae of Bombyx has been found by Kobayashi and co-investigators (83) for progesterone, hexestrol, diethyl-stilbestrol, cholestanol, 7-dehydrocholesterol, campesterol,and noradrenalin.

Periodicity in insects is explained by external stimulisuch as a change in temperature (82, 121), feedings (118),etc. (81) activating the brain to secrete. Apparentlylow temperature does not induce imaginai differentiationof Dauer-pupae (76). In Platysamia, vanderKloot (112)showed that the brain, which is dormant and electricallysilent during the winter, produces cholinesterase and showselectrical activity at the time neurosecretion appears.This is then followed by changes in morphology of theprothoracic gland. Transplantation experiments indicate that larval brain is as effective as pupal brain inaffecting the imaginai differentiation of Dauer-pupae (71).

Ecdysones.â€”The prothoracic gland in lepidoptera is amore favorable object for ablation and transplantationthan corresponding cells in diptera where they lie inproximity to cells of the corpora aliata to form the ringgland. From experiments of this type it is apparent thatsuccessive larval molts are brought about by balancesbetween levels of hormone from corpora aliata and pro-thoracic glands, and transition between larval and pupaland pupal and imaginai (adult) stages are induced byhormone from the prothoracic glands. Extraction of thehormone of the prothoracic glands is based on pioneerwork of Becker and Plagge (6) on isolating active material.Active hormone was first crystallized from chrysalides ofthe silkworm by Butenandt and Karlson (31), who namedit ecdysone. This water-soluble material has the sameaction as the secretion from transplanted prothoracicglands. Recently, Karlson (65) has published a tentativeand partial formula for the ecdysone he has isolated. We(23) have obtained five separate, active fractions of ecdysone from Bombyx, two (a and /3) previously reported byButenandt and Karlson and three (7, 5, and e) previouslynot isolated. The relationship of the latter to the chemical structure reported by Karlson and to the activity ofthe gland is not currently known. The likelihood thatthese are steroids with cholesterol a precursor is great.

We (20) have carefully followed the levels of ecdysonesin Bombyx during various stages in the life cycle and have

Â°=C.U./6.Wet Weight

â€¢=C U/Individuo!

JF-G-LPP I 2 3 4 5 6 7 8 9 IO II 12

Larvalâ€”â€¢+* Pupal H* Imaginaiâ€”LEVELS OF ECDYSONEDURINGMETAMORPHOSIS STAGES

CHART 1.â€”Levels of ecdysones during metamorphosis ofBombyx mori.

found increased titer before pupation, with precipitousfall and gradual rise thereafter (Chart 1). Samples fromfull-grown larvae, silkworms in the prepupal stage, andsilkworms at 1, 2, 3-4, 5-6, and 6-7 days of age were usedin the tests and extracts bioassayed by means of theCalliphora test. The prothoracic glands from which thefractions we have isolated presumably originate undergodissolution soon after the adult stage is reached.

Apparently ecdysones are the substances responsible(39) for eliciting puffs (89) on the chromosomes1 whichBecker (5, 6) discovered to appear in response to hormonefrom the ring gland. Recently Kroeger2 has found thatseveral simple substances, including ZnCl2, chloroform,butanol, and urethan, mimic action of ecdysones onchromosomes. He has also proposed that successiveNa+:K+ ratios activate respective genetic loci and thatecdysones act by increasing the concentration of K+ ionsin the nuclear "sap" whereas juvenile hormone is effectiveby maintaining a high level of Na+ ions.

Juvenile hormone.â€”Wigglesworth established some yearsago (116) that the corpora aliata are the source of juvenilehormone. This hormone is a mandatory component ofthe mechanism to induce larval molting. It acts in opposition to ecdysone in the direction of retaining lessmature characteristics of the organism. Although thisis probably not a simple inhibitory effect (118), its exactmechanism remains speculative (56). Kobayashi andBurdette (74) found that heterologous transplantationof corpora aliata between lepidoptera diapausing in eggand pupal stages resulted in different effectiveness in aforeign environment. Also similar transplantations (60,75) suggest that the corpora aliata may act in pupal andin imaginai differentiation under certain circumstances toretard differentiation3.

Large quantities of the hormone (49, 54) are present inthe abdomen of adult male Hyalophora (Platysamia)

1W. J. Burdette and R. Anderson, Sequence of Puffing inSalivary Glands Following Administration of Ecdysones, unpublished.

! H. Kroeger, personal communication.Â»M. Kobayashi and W. J. Burdette, Histophysiologic Studies

on the Corpora Aliata in Dauer Pupae of the Silkworm, Bombyxmori, to be published.

on June 14, 2021. © 1964 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from


BuRDETTEâ€”Invertebrate Hormones and Differentiation 523

cecropia (122-124). Also, Gilbert and Schneiderman(51, 52, 105) and Williams and co-workers (124) isolatedmaterial from a number of diverse biological sourcesincluding bacteria and vertebrate adrenal cortex andthymus that gave a positive bioassay for juvenile hormone activity. Subsequently, Schmialek (104), notingthat similar active material could be isolated from thefeces of Tenebrio, was successful in extracting farnesoland its oxidation product, farnesal, from this source.Wigglesworth confirmed that these were active on bio-assay. However, the activity of farnesol is not greatenough for one to assume that this is identical with thenatural hormone. Farnesyl phosphate is also not veryactive, but the methyl ether of farnesol and N, N-diethyl-farnesyl amine are very active (65). Schneiderman et al.also find a number of compounds including phytol,linaloÃ¶l,solanesol, and nerolidol to have activity similarto juvenile hormone on bioassay. The intermediateposition of farnesol in the metabolism of cholesterol is ofinterest in view of the report of Kobayashi that cholesterolis the product of neurosecretion of the brain and isprobably a precursor of the ecdysones.

In the adult, the corpora aliata secrete a substancenecessary for the formation of the yolk (92, 110, 116).Also, Schneiderman has suggested that secretion fromcorpora aliata may stimulate the prothoracic gland sinceKrishnakumaran and he found that farnesol can activatethe prothoracic glands of A. polyphemus.

Bioassay.â€”Several bioassays have been useful in thecourse of investigations on hormonal control of metamorphosis. Pupae with brains ablated (Dauer-pupae)have been used to test the effect of hormone extractedfrom the brain (50). The cuticle of various species ofmoths (50, 55, 106) as well as Tenebrio have been used inthe bioassay of juvenile hormone; and Calliphora, theblow fly, has been used as a means for detecting ecdysone.

The Tenebrio test (65) is carried out by injecting 0.5pi. of the substance to be assayed into the abdominal wallof pupae of meal beetles when they are 24-48 hours old.Activity for juvenile hormone is present if a portion of thecuticle has the pale color characteristic of the pupal stageinstead of the dark brown appearance characteristic ofthe adult when the beetles emerge in 8-10 days.

The bioassay for ecdysone is carried out by ligatinglarvae of this carnivorous fly posterior to the ring gland.If this has been done at the appropriate stage, the anteriorportion pupates, and the posterior portion remains in thelarval stage. The latter is then injected with the extractto be tested. A positive test consists of pupation within24-48 hours. A weighted average is used, and 50 percent pupation of 20 posterior segments or more constitutes a positive test. The test is essentially a refinementby Karlson of that developed by Becker and Plagge (4),based in turn on earlier work by Fraenkel.

Metamorphosis.â€”The holometabolous insects havelarval instars followed by transition from larva to pupaand then emergence from pupa to adult. The adult stageis entered from the last larval instar in the hemimetabolousgroup. Information about the control (14, 15) of thesetypes of differentiation has come principally from experiments in which various glands have either been ablated or

transplanted or a combination of the two used. Ligationhas also been used to impede the distribution of hormone.To recapitulate events in metamorphosis (Chart 2), thebrain becomes electrically active as a result of some external stimulus, such as a change in temperature; neurosecretion occurs; and the prothoracic glands then becomeactive to secrete the ecdysones. This is reminiscent of theanalogous tropic action of the hypophysis and the glandsresponsive to it in vertebrates (102, 103). The balancebetween the amount of ecdysones and juvenile hormones(possibly in the form of congener|s] of farnesol from corpora aliata) acts on sensitive tissue in such a way as toregulate metamorphosis from one larval instar to the next.Continued action of ecdysones brings about differentiationfrom the last larval instar onward. Hormone from thecorpora aliata may also play a role in imaginai differentiation.3 In addition to the hormonal mechanics discussedpreviously, the subesophageal ganglion of the silk mothproduces a hormone that regulates the egg diapause, andfemales given injections of extracts are induced to laywinter eggs (59).4

The significance of positive bioassay for brain hormoneand juvenile hormone in the case of a large number ofsteroids, alcohols, and other substances known in vertebrates (13) is difficult to assess. Whether the bioassay issimply not very specific or there is some similarity instructure and/or action between them and natural hormones is open to question. We have assayed a number ofwater-soluble steroids by the Calliphora test and so farhave obtained negative or equivocal results. The chemical nature of those hormones related to metamorphosisthat have been studied most thoroughly are cholesterol(79), a precursor of cholesterol (104), or a possible derivative (66). Sterols are essential components of the diet ofinsects, since biosynthesis otherwise proceeds no fartherthan squalene. Although cholesterol may be an activecomponent of the brain and may act by stimulating secretion of the prothoracic gland in addition to providing themolecule from which ecdysones are elaborated; althoughecdysone with formula similar to that published by Karl-son may be secreted by the prothoracic glands; and although a substance structurally similar to farnesol maybe produced by the corpora aliata, in every case there isreasonable likelihood that other components of the secretory product are active as hormones. In addition, thespecter that the biological action described for each hormone is rather nonspecific has been raised by the extensivedistribution of material having brain- and juvenile-hormone activity in nature and the report that rather simplecompounds have an action on chromosomes similar tothat of ecdysones. Nevertheless, the chemical storyemerging is a rather coherent one, and for the first timean experimental approach for probing differentiation inchemical terms is becoming available.

TUMORS IN DROSOPHILA

It is of interest to determine how the regulatory systemsdescribed affect atypical growth in invertebrates. The

4Other neurohormones extracted by Cameron (33), Carlisle(34), and Gersch et al. (48) affect muscular contraction.



BRAIN

-CH2 -C = CH-CH2 -CH2 -C--CH-CH2 OH

NEOTENIN(S)

LARVALINSTARS

IMAGO

METAMORPHOSISCHART2.â€”Diagram of the interaction of hormones from brain, corpora aliata, and prothoracic

glands during metamorphosis.

524




tumors that appear in Drosophila (19) usually consist ofaggregations of cells that do not divide or divide veryseldom. They appear early in larval life and undergodissolution later. Pigmentation of the inert residuumremains throughout the remainder of the life cycle andprovides a convenient marker of those animals that havehad tumors in the larval stage. As a rule, the processdoes not interfere with the viability of the individualsaffected. The phenomenon is inherited, and more than 50strains have been described. The genes responsible forsusceptibility are present on from one to four chromosomesand consist of main genes, suppressors, and enhancers invarious combinations. For some reason, more are locatedon the second chromosome, but few are allelomorphic.The mutants that have been examined show no evidenceof position effect and are point mutations, most of whichhave occurred spontaneously.

The effect of the hormonal system regulating metamorphosis on these tumors has been tried in two ways:first, by ligating larvae posterior to brain and ring gland(17); and, second, by introducing a gene on the secondchromosome (18) that arrests development, resulting ingiant larvae due to a congenital defect of the ring glandwhich contains cells analogous to those both in the corporacardiaca and prothoracic glands of lepidoptera and otherinvertebrates. When hormone from the ring gland isexcluded by either means, the number of individuals withtumors is greater than in those with normal glands, indicating that there is a release of hormonal inhibition.This has been confirmed by Oster (89).

HORMONAL HETEROPHYLYExperiments in vivo.â€”Since the processes that have

been described in insects constitute a powerful regulatorysystem for differentiation and the control of growth whichis distributed widely throughout the animal kingdomincluding not only diptera and lepidoptera but also crustaceans and other invertebrates, the possibility that thesematerials may have an effect on mammalian tissue seemedworthy of scrutiny. The place of at least one of the activecomponents produced by the brain (cholesterol) and of thecorpora aliata (farnesol) in mammalian metabolism isalready well known. On the other hand, we extracted anumber of human tissues and failed to obtain a positivebioassay of the product for ecdysone (22). The portionof the formula reported by Karlson also fails to suggest thatit occurs naturally in mammals, although structurally it isrelated to bile acids and some of the other steroids knownto be important in mammalian metabolism. It shouldbe noted, however, that other components secreted byboth brain and corpora aliata may be important in theprocess of differentiation and yet remain undiscovered.The additional ecdysones we have recently described areexamples.

The first experiment was performed by injecting respective active extracts of brain hormone, juvenile hormone,and ecdysones into animals bearing transplanted tumors.Sarcoma 180 was transplanted into C3H mice, and because of the small amount of brain hormone availableonly a few mice were given injections. The negativeresults are understandable if the principal activity is due

TABLE 1EFFECT OF FARNESOL ON SARCOMA180

DOSE (ml.)PERINJECTIONControl.05.10.10.10.20.20NO.INJECTIONS353131No.

DEATHSWITHIN48007111313NO.

OrREGRESSIONSNone509107679Partial2144337Complete30135016PERCENTAGE

REGRESSION9103350573072

A-strain mice were given inoculations subcutaneoualy at 60days of age of a small (1 mm.) fragment of Sarcoma 180. Whenthe tumor was established and growing, farnesol was injectedsubcutaneously in the opposite flank in the respective scheduleof dosages given in the table. Observations were made dailyand weight of the animals and measurements of the tumorsrecorded at least twice each week thereafter.

to cholesterol (79). Various dosages of ecdysone wereinjected intraperitoneally into mice bearing transplantedtumors; and, although there was suggestive regression of21 per cent of the tumors, this was not proportional to thedosage (21).

Considerable toxicity was attendant on the injection offarnesol. As high as 72 per cent of the tumors regressedeither completely or partially when up to 0.2 ml. wasinjected (21)5 (Table 1). Divided doses did not seem toreduce the toxicity in all cases, and there was some lossin the cancerocidal effect by giving the material in thismanner.

Since juvenile hormone tends to perpetuate larvalcharacteristics and possibly inhibit differentiation, it wasthought that it might be teratogenic in mammals. Sofar results of investigations on this point are negative.6Farnesol injected into pregnant mice failed to induce morelethals in utero than were found in mice without treatment,and no malformations were found in the offspring of thosefemales allowed to reach parturition.

Experiments in vitro.â€”From these experiments itseemed likely that pursuit of the problem with biologicalsystems in vitro would be more rewarding and that theeffect suggested by ecdysone would be most likely to showspecific effects on mammalian tissue. Therefore, bothembryonic fibroblasts and Sarcoma 180 were cultured inhanging-drop cultures, and the effect of ecdysones wastested. The degree of growth was scored uniformly,and it was found that the growth of embryonic fibroblastsand neoplastic tissue was inhibited (25). The sequence ofinhibition was somewhat different, however, when thegrowth of the two tissues was compared. In the case ofembryonic fibroblasts, increasing concentrations of ecdysone progressively delayed the onset of growth. On theother hand, Sarcoma 180 began to grow and persisted fora time inversely related to the strength of the dose.

6W. J. Burdette, Effect of Farnesol on the Growth of Sarcoma180, unpublished.

â€¢W. J. Burdette, J. Simmons, and R. Anderson, The LethalMutation Rate in Mice Following the Administration of Farnesol,unpublished.




TABLE 2EFFECT OF ECDYSONES*ON PROLIFERATION OF HELA CELLS!

IN VITRO

TABLE 3EFFECT OF ECDYSONESON HELA CELLS

DOSEcu./ml.t100500100015002000GROUPControlTestControlTestControlTestControlTestControlTestINOCULUMJ3.13.10.40.41.01.00.70.70.90.9DAYÂ§410.910.51.41.53.93.41.00.91.51.4s11.410.72.32.35.54.41.61.52.81.5612.311.05.43.53.94.5DeadDead78.710.33.72.55.34.5g6.56.73.22.64.23.3

â€¢Hormone added on day 3.t Cells grown in Eagle's minimal essential medium with calf

serum.Ã•Calliphora units/ml.Â§Number of cells X IO6 (counted with Coulter counter after

trypsinization).

In order to obtain less subjective evaluation of changesin rates of proliferation, a Coulter counter was used todetermine the number of HeLa cells in culture, and theexperiment was repeated.7 Cultures with a known number of cells in suspension were treated with ecdysone onthe 3d day of growth. Aliquots of the cells in suspensionwere then counted, and the mean size of the cells wasdetermined on successive days after the cells had beentreated with trypsin. It was found (Table 2) that increasing dosage progressively shortened the time of proliferation and the number of cells in the culture, and athreshold of dosage was found below which the hormonewas not effective in inhibiting growth. The same type ofexperiment was performed utilizing E. coli and Staphylo-coccus aureus.* In the concentrations of hormone used,there was no significant alteration in the growth of thecultures when hormone was added during the log phase ofgrowth. The amount of DNA and RNA in HeLa cellscultured with and without ecdysones was determined, andan increase was found in both when means were compared9(Table 3). These and the experiments described subsequently were all carried out with ecdysone extractscarefully bioassayed.

Metabolic studies.â€”Since the addition of ecdysone toRhodnius results in the proliferation of mitochondria anda change in their gross appearance under the light micro-scrope according to Wiggelsworth (119), tissue was selectedthat is adapted to aerobic metabolism and known to have

7 W. J. Burdette, Effect of Ecdysones on the Growth of HeLaCells in Tissue Culture, unpublished.

â€¢W. J. Burdette, The Growth of Bacteria in Cultures Containing Ecdysones, unpublished.

â€¢W. J. Burdette, DNA and RNA of HeLa Cells Cultured inMedium Containing Ecdvsones. unpublished.

ControlEcdysones*DXAtWg/cell1629pg/mg20.087.8RNAtWig/cell6582Mg/mg70.1167.6

* 600 Calliphora units/ml,

t Diphenylamine method.ÃŽOrcinol method.

a huge number of active mitochondria in order to test theeffect of ecdysone on aerobic respiration. Slices ofmammalian cardiac muscle were placed in Warburg vessels,and Qo2 was determined under various conditions withand without the addition of ecdysones (21). There wasa small but suggestive increase in Qo2 when the exogenoussubstrate used was lactic acid. Otherwise the ecdysoneshave not given any changes of note so far, and mitochondria of mammalian liver are unchanged in appearanceunder the electron microscope during hepatic perfusionwith ecdysones.8

Protein synthesis (118, 126).â€”It also seemed desirableto determine the effect of ecdysones on protein synthesisin the cytoplasm. A fraction of mammalian liver obtained by centrifugation of homogenates at 14,000 Xg was incubated with hormone, and leucine labeled inposition one with carbon 14 was added. The fine structureof this fraction,9 sedimented at 20,000 X g and recognizable with the electron microscope, consisted of smoothand rough endoplasmic reticulum, glycogen, and a fewintact mitochondria (Fig. 1). The incorporation intoprotein from zero time was determined by plating thematerial precipitated by trichloroacetic acid and countingin the proportional range. These results were comparedwith those in preparations without hormone. There wasa significant increase in the incorporation of leucine intoprotein when 250 Calliphora units of ecdysone per ml. ormore were used (24). Although there was a disturbingvariation in the uptake from one sample to the next,incorporation increased in every instance in those dosageswhen sufficient hormone was present in the preparation.However, it is noted that there is apparently a maximummean beyond which the uptake did not progress and thatthe control varied more than the experimental group. Thesignificance of this is not known. The limitations of sucha system are well known, and the fact that messengerRNA is unstable (113) in such a situation has been documented hi studies on microorganisms. However, thefact that there was an increase is suggestive.

Ultrastructure of mammalian liver perfused withecdysone.â„¢â€”Sinceresults in vitro with the ribosomal fraction of mammalian liver suggest enhancement of the rateof protein synthesis when ecdysones are added and themorphology and rate of growth of mammalian cells arealtered by extracts containing the hormone, the ultra-structure of mammalian tissue perfused with ecdysone

10W. J. Burdette and T. P. Ashford, Fine Structure of Mammalian Liver Following Perfusion with Ecdysones, unpublished.




has also been studied. Livers from fasted rats wereperfused with blood containing ecdysone, and sampleswere taken at intervals of 10 minutes. The fine structurewas then compared with that in sections from liver perfused without hormone. Preliminary observations revealthe following picture after perfusion (Fig. 2): coarsegranules are prominent in nucleoli; large cytoplasmicvacuoles are present; the rough endoplasmic reticulum isprominent and is usually arranged centrally in the cytoplasm; the mitochondria are aligned with the roughendoplasmic reticulum in the usual fashion; the smoothendoplasmic reticulum is distributed diffusely throughoutthe cytoplasm; and pinocytosis at the cellular surface iseasily seen.

Organization of the rough endoplasmic reticulum (Fig.3) in chemical terms may take the form illustrated in thediagram (Chart 3), since polypeptide synthesis from activated amino acids takes place through the interaction ofmessenger RNA, aminoacyl sRNA, and heavy ribosomes(109, 113). That ecdysones may have a direct effect onthis organization rather than a secondary effect as a resultof its activity on the chromosome is suggested by theevidence that ribosomal fractions incubated in vitro without intact chromosomes incorporate leucine into proteinat an increased rate as well as the morphologic appearancementioned. In some of the preparations from fastedanimals perfused with ecdysones the rough endoplasmicreticulum was organized and prominent, reminiscent ofthe picture in the liver of animals that were fed.

The pinocytosis observed at the cellular surface suggestsample transport at the periphery of the cell. The ideathat this is facilitated in primary or secondaiy fashion byaction of ecdysones is attractive but has not been proved.Whether the large vacuoles observed represent a toxiceffect such as that observed with hypoxia or whether thisrepresents transport of product from the cell or ingressand accumulation of substrate from without awaitsfurther study. These vacuoles are the most consistentdifference between liver from fasted rats perfused withecdysones and those without addition of extract to perfu-sate.

These observations of mammalian hepatic fine structureafter perfusion with ecdysones provides an interestingcomparison to the effect of the same substances on thearchitecture of the cell in insects. Wigglesworth (118)noted enlarged nuclei and nucleoli, increased RNA between the nuclei, increased size and number of mitochondria, large amount of protein, and "highly developedand often lamellar ergastoplasm" in the cells of Rhodnius

following the administration of ecdysone. Cells of thefat body appeared to have a lower threshold for the actionof ecdysone than did other tissues studied in decapitationexperiments. The similarity between the changes inducedby ecdysone and those appearing as a result of injury toinsects continues to be intriguing. (We11 have found

that the tensile strength of mammalian wounds is notenhanced by extracts containing ecdysones, however.)Wigglesworth has suggested that ecdysone may influence

" W. J. Burdette and R. Price, The Effect of Ecdysones onHealing of Mammalian Wounds, unpublished.

intraccllular permeabilities in insects so as to allow accessof enzyme to substrate for the assimilation of amino acids.He does not regard the action of neotenin as inhibitorybut as a substance reacting with gene-controlled enzymaticsystems to provide the realization of a choice of alternativemorphologic forms.

CHROMOSOMAL RESPONSE TO ECDYSONES

Very little direct information is available on the stateof the mammalian chromosome during interphase or earlyprophase. Three special cases have attracted attention asvisible structures from which inferences applicable to lessfavorable material may be drawn concerning chromosomalstructure and function. They are the spiral structuresobserved in amoebae (94), the lampbrush chromosomesin oÃ¶cytesof certain amphibia, and the dipteran salivarychromosomes (1, 65, 93, 107). The significance of thefirst of these remains controversial. In the case of lamp-brush chromosomes, peculiar loops extend outward fromthe chromosomes of these leptotene structures. Significant amounts of RNA are present in the loops, and oneside is thicker than the other. A prodigious amount ofstudy has been devoted to the prophase chromosomes ofthe salivary glands of insects, since the significance ofthese structures was first recognized by Painter (90).Similar structures are seen in the midgut, rectum, testes,and malpighian tubules but have not been examined as

RNAC=0(de)'-^~p^

i ^V"NRNA-A

A,

Â¿


extensively. The salivary chromosomes in Drosophila,Chironomus, and Rhynchosciara have been favoriteobjects for cytologie study for many years.

The salivary chromosomes consist of parallel duplications (99) of the paired somatic chromosomes so that 64,128, or more lie side by side with the matching chromo-mÃ¨resgiving the large, elongated structures a bandedappearance. These bands are constant in their interrelationships and morphology and may be used to identifyspecific regions. Chromosomal aberrations have beenuseful in identifying the location of mutants, most of whichcause no visible structural alteration. The bands areFeulgen-positive and contain DNA.

Painter was the first to note knoblike enlargements orpuffs on the salivary chromosomes. These puffs appearquickly in the salivary chromosomes and regress rapidlyafter a characteristic period of persistence. Changes inthe pattern of puffing are correlated with the progressionof events during the life cycle and presumably are somehow a reflection of genie action responsible for the changesknown as metamorphosis. Becker (5) has carefullyrecorded the temporal-morphologic relationships of puffingin Drosophila melanogaster and is responsible for establishing that the hormone of the cells in the ring gland corresponding to prothoracic gland is responsible for elicitingthe puffs. He was able to identify approximately 70regions showing distinct puffing on the X, second, andthird chromosomes. Glands transferred to Ringer solution for a short period of time regressed to an earlier pattern of puffing when explantation was carried out beforea certain critical period in the life cycle. Later, development toward a more advanced pattern of puffing occurredin vitro. Also puffing was observed in vitro that did notoccur at that site in the intact animal. When the l(2)glgene is homozygous, the normal puffing pattern is derangedso that no puffs characteristic of the corresponding phaseof larval development are found. Different types ofcells in the same or different organs may have a differentpattern of chromosomal puffing. Even though the patternmay be similar in the same tissue, we have often observedsalivary cells side by side presenting different stages of thesame pattern so that the synchrony is obviously imperfect.Also Becker (5) noted a puff in the anterior portion of thesalivary glands of Drosophila not appearing farther posteriorly. Kroeger (86) reported the induction of a different pattern of puffing in the chromosomes of Drosophilawhen the salivary-gland nucleus was transplanted intothe cytoplasm of the egg.

Many investigators have been interested in the chemicalcomponents and their concentration in the puffs (95,97, 105). Recently Edstrom and Beermann (11, 42)concluded from studies on the RNA of single giant puffsthat this RNA is different from that in the nucleolus andfrom most of that in the cytoplasm. During the firsthalf hour after injection of tritiated uridine and otherprecursors of RNA, the puffs are labeled more heavilythan any other part of the cell according to Felling (91).Rudkin (95, 98, 99) found a consistent but not significant increase of RNA formation concomitant withformation of a specific puff in Drosophila melanogaster, andRudkin and Corlette (98) found larger amounts of DNA

(108) in puffs than elsewhere during the prepupal stage.Earlier, Breuer and Pavan (16) reported an increase inFeulgen staining in puffs of Rhynchosciara. Labeledcytidine but not thymidine was incorporated to a greaterextent into puff 60B of Drosophila than elsewhere, according to Rudkin and Woods (100). Breuer and Pavan(16) found indications that disproportionate DNA synthesis occurred in puffs in Rhynchosciara, and the capacity to synthesize RNA and the amount of DNA containedwas found by Stich and Naylor (110) to be variable inpuffed regions of the salivary chromosomes of one of theChironomids. Therefore the chemical composition ofpuffs varies from one to another, may not be the samewhen identical regions are compared in different tissues,and may vary from one stage to another in the life cycle.The puffs showed no consistent response to treatmentwith an analog, amethopterin, expected to alter metabolism of nucleic acid in experiments by Corlette (40).There was also no influence of colchicine on these structures. These and other observations have tended tofocus attention on the RNA content of the puffs, although their exact chemical composition is difficult todetermine because it is not constant and because of deficiencies in methods of analysis.

A type of secretion granule unique to a small region ofthe salivary gland in Chironomus was found by Beermann(9) to be due to a genetic factor located at the locus of apuff on the fourth chromosome, appearing only when thesecretion granules appeared. He looks upon this as anexample of the cellular specificity of the puff and thefunction (7, 8) it controls. It is not clear why puffs andgranules do not occur in the remainder of the salivarygland bearing the gene in question.

As an extension of the observation that the defectivering gland associated with the l(2)gl gene results inchromosomes without puffs, the results of injections withecdysones are of interest. Clever and Karlson (36, 37,39) observed that puffing in the salivary chromosomes ofChironomus is enhanced in certain regions, and the intensity of the effect depends on the dosage of hormone(38). This effect appears within 30-60 minutes. Asecondary sequence of puffing then appears 6-48 hourslater, also related to dosage. Therefore, as one wouldexpect from projecting the results of Becker, ecdysonesare the substances responsible for the effect of secretion ofprothoracic glands on the puffing pattern of the salivarychromosomes.

In our laboratory the appearance of salivary chromosomes in Drosophila (Fig. 4) after injection of ecdysonesor culturing the glands in saline containing ecdysones hasbeen changed.1 The size of the puffs has been increased,sometimes to gigantic size with margins fading into thecytoplasm, suggesting a release of material from the regionof the puff. So far only regions forming these enlargements in the ordinary course of development have beenobserved to do so following treatment. Larger doses ofecdysones are probably required to produce the effect inDrosophila than in Chironomus, and the effect of hormoneappears suddenly in approximately 20 minutes. When apuff has a different appearance in pupal and larval stages,ecdysones injected during larval instars may produce a




puff with pupal characteristics. Large doses of hormonecause the chromosomes to swell and apparently to assumemore adhesive qualities. The clear areas between bandsbecome somewhat distended, but the width of the chromosomes is not changed appreciably.

An examination of mutants in or about the region of thepuffs in Drosophila melanogasler fails to give any uniformpicture of a type of genie action or adaptation to thespecific function of the tissue in which the puffs appear.This is not particularly surprising, since genes situated inclose proximity may have very different action, and thegenie action signified by appearance of a puff may occurat a time remote from the action responsible for the mutants listed and detected in the adult. In Drosophila athreshold of dosage exists, but otherwise the appearanceof the chromosomes is not changed by alterations in dosageof hormone unless exceedingly large amounts are used.Theoretically the locus where the puffs appear representsthe site of increased genie activity, and the genes concerned in events taking place at the time during the lifecycle when they appear are located in the same regionsof the chromosomes.

INVERTEBRATE HORMONES ANDDIFFERENTIATION

The current status of knowledge concerning events inmetamorphosis of insects (56, 63, 64, 88) may be combined with evidence from mammalian and microbialsystems (2) to construct a hypothesis applying to thegeneral process of differentiation, if license is permittedto overlook certain deficiencies in current information.A balance between the effect of neotenin and the ecdysonesis apparently responsible for a number of the events inmetamorphosis of insects. The concentration of eachhormone in relation to the other affects larval and adultcells in such a fashion that an orderly process of growth,replacement, and differentiation ensues. How this isaccomplished at the cellular and molecular level is moreapparent for the ecdysones. Also this group of hormonesprobably has effects that are more than nonspecific onmammalian cells, so that the influence of invertebratehormones on molecular events may have some generalsignificance.

The ecdysones activate sensitive loci on the chromosomes and possibly increase the rate of protein synthesisin the cytoplasm. The question whether the materialselectively produced as a result of the action of ecdysonesis exclusively messenger RNA (10) or other substances aswell requires much additional work. Also whether theyincrease transport at the surface of the cell is the subjectof current investigation. These mechanisms obviouslyenhance synthesis of proteins of specific type, some ofwhich in turn may be regulatory. The effect on thechromosome as well as elsewhere may constitute a mechanism of release such as increased permeability or removalof inhibition. An inductive activation of enzymes (62)could also be involved.

These observations yield one example of some of thesteps in differentiation in molecular and morphologicterms and suggest how the cellular product, a hormonein this case, may constitute a feedback mechanism selec

tive for individual opÃ©ronson the chromosome. Itemphasizes the importance of the sequence of events, butit does not divulge general secrets of irreversibility andfails to give the ultimate answer about why the templateis activated or sensitive at one chromosomal locus and notat others. Evidently the explanation for different stepsin differentiation must be solved in terms of individualmolecules, chemical reactions, genes, cistrons, opÃ©rons,etc. Invertebrate material offers advantages in bridgingthe mÃ©thodologiegap between microbiologie and mammalian systems. Retrogressions so obvious in malignantcells should be understood the better as a result of suchan approach.

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FIG. 1.â€”Ultrastructure of homogenate of liver from the ratused to test effect of ecdysones on the rate of incorporation ofleucine into protein. Smooth endoplasmic reticulum and gly-cogen are easily seen. The latter obscures the rough endoplasmicreticulum. Occasional mitochondria are also present.

FIG. 2.â€”Hepatic cell of the rat following perfusion with ecdysones. (N: nucleus; n: nucleolus; V: vacuole; RE R: roughendoplasmic reticulum; M : mitochondrium)



Fio. 3.â€”Rough endoplasrnic reticuluni (RER) of liver (rat)after perfusion with ecdysones.

Fio. 4.â€”Puff (65) on chromosome III L of Drosophila melano-gaster. (Puff is in center of plate.)

532



R ER

533




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1964;24:521-536. Cancer Res Walter J. Burdette DifferentiationThe Significance of Invertebrate Hormones in Relation to

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CANCER RESEARCH...cuticles in Rhodnius prolixus, and Fraenkel (43) reported similar findings for Calliphora. Weyer (115) demon-*Aided by a grant from the Department of Health, Education,