a study of carbohydrate metabolism in planorbis …2015/12/01 · embryol. exp. morph., vol. 12,...

/ . Embryol. exp. Morph., Vol. 12, Part 1, pp. 15-26, March 1964Printed in Great Britain

A study of carbohydrate metabolismin Planorbis exustus

by GAJANAN V. SHERBET and M. S. LAKSHMI1

From the Department of Zoology, University ofPoona

INTRODUCTION

T H E processes of embryogenesis, namely differentiation and growth, and alsothe maintenance of the organism, require energy. Needham (1950) claimed thatduring development carbohydrate, protein and fat metabolism came into opera-tion in that order. This sequence may differ in some species (Levtrup, 1959a, b).The importance of -SH groups in the morphogenesis of Planorbis exustus wasearlier studied using chloroacetophenone (CAP), an -SH inhibitor, which causedarrest of development, delay in cleavage, exogastrulation etc. when cleavage-stage embryos were treated (Mulherkar & Sherbet, 1963). Since -SH reactants arealso inhibitors of carbohydrate metabolism, we felt it desirable to find out ifcarbohydrate metabolism preponderates during the cleavage period and therebydecide whether the action of CAP is to inhibit this metabolism or to interfere withthe -SH groups taking part in the formation of the mitotic spindle, regarding whichRapkine put forward the theory of reversible denaturation. We have, therefore,undertaken a study of the metabolism of P. exustus using sodium azide and iodo-acetamide (IAA). There is reason to believe that the development of this molluschas different kinds of metabolism dominant in its different phases of development.

MATERIALS AND METHODS

The egg masses of P. exustus were detached from the undersurface ofNymphaealeaves and were treated with sodium azide at concentrations of 0-001,0-002 and0-003 M (Series A, B and C) for 1 hr. or indefinitely. In some experiments theegg masses were returned to normal pond water to remove the block to develop-ment. Iodoacetamide, in concentrations of 0 • 001,0 • 0005,0 • 00025 and 0 • 0001 M,was the other inhibitor employed (Series D). Proper controls were maintainedin all the experiments. The method of treatment was the same as that followed byMulherkar & Sherbet (1963). The control and experimental egg masses wereallowed to grow in standard glass Petri dishes (50 mm. diameter). Not more than

1 Authors' address: The Department of Zoology, University of Poona, Poona 7, India.

16 G. V. SHERBET and M. S. LAKSHMI

ten embryos were placed in each Petri dish containing 10 ml. of the control orexperimental solution. The development of the embryos was carefully recordeduntil they succumbed to the treatment or hatched. The text-figures were drawnafter narcotizing the animals with menthol. The animals were fixed in Bouin'sfluid, serially sectioned at 10 /x and stained by iron-haematoxylin.

EXPERIMENTAL RESULTS

In these experiments 1033 embryos were treated with azide and IAA with 613embryos serving as controls. The results of the experiments with azide (continuoustreatment) are given in Table 1.

A few words of explanation of the data given are required.

Mortality

The mortality of experimental embryos given in column 6 refers only to deathsoccurring before any specific abnormal development or malformation was noticed.This does not include the total mortality observed by the end of the experiments.For example, in some experiments involving cleavage-stage and gastrulatingembryos, gastrulation stopped and the embryos became hydropic, all ultimatelydying without further differentiation, but this has not been considered as 100 percent, mortality.

Vesiculation

Under this head we have included embryos grown beyond 72 hr. which haveenlarged and become transparent due to accumulation of fluid. Such accumula-tion of fluid took place in the region posterior to the cephalic ganglia, in thecephalic region, in the foot or all over the organism.

Hydropia

This abnormality occurred on treating gastrulating embryos with azide orwhen those already in azide reached the gastrula stage. Normally, in such cases,gastrulation stops halfway, so that the archenteric cavity does not fill the blasto-coel and the embryos become enlarged and transparent with a mass of cells in thecentre. Text-fig. 1A represents a section of hydropic embryo produced by treat-ment of 3rd-cleavage embryos with 0-001M azide. Text-fig. IB is a section of anormal embryo (approximately 48 hr. old) and is intended for comparison withText-fig. 1A. It should be noted that in the normal embryo the differentiatedalbumen cells completely fill the interior of the embryo, but that in the hydropicembryo though these cells are differentiated they do not fill up its interior. Wewould like ito mention here that the albumen cells of hydropic embryos differ instructure from those of the normal embryos in that the large albumen vacuoles

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TEXT-FIG. 1 A: Transverse section of a hydropic embryo (72 hr. old) produced by continuoustreatment with 0 • 001 M sodium azide of 3rd cleavage-stage embryos. The large space in theinterior of the embryo and the structure of the albumen cells (alb. c.) to be noted. Arch, isthe archenteric cavity.

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B: Transverse section of an approximately 48-hr, old normal embryo, for comparisonwith Text-fig. 1A.

Carbohydrate metabolism in Pianorbis exustus 19

are present at the pole of the cell which is away from the archenteron in the normalembryo whereas in the hydropic embryo these albumen vacuoles are situated at thearchenteron end of the cells.

Shell malformation

This included disproportionate shells or those which were very small andremained as a cap on the visceral hump. Embryos which lacked shells altogetherhave also been included under this head.

Series A experiments (sodium azide 0 • 001 M)

Effect on cleavage-stage embryosA 15 min. treatment of some of the cleavage stages (up to approximately 6-hr.-

old embryos) did not produce any effect on the development and the experimentalembryos hatched as normally as did the controls. The cleavage stages were there-fore given 1 hr. or continuous treatment in view of the observation of Hall &Moog (1947) that the developmental block produced by azide is reversible. It wasobserved that at whatever stage the treatment commenced the eggs cleaved nor-mally until they reached the gastrula stage. The embryos did not develop further,however, they became hydropic and finally degenerated when allowed to remainin azide in this arrested state of development. It is interesting to note that therewas no delay in cleavage and at each step the cleavage divisions in the experimen-tal embryos coincided with those in the controls. This means that the embryoscontinued their development without inhibition for 18-24 hr. despite the presenceof azide in the surrounding medium.

Effect on 24- and 48-hr.-old embryosContinuous treatment of 24-hr.-old embryos caused them not to develop at all,

to become hydropic and finally to degenerate. Embryos 48-hr, old showed somedevelopment, unlike the 24-hr.-old ones. But it was retarded from 50-108 hr. ascompared to that of control embryos. Hydropia was of usual occurrence, andwhenever it occurred there was no further development. If development did takeplace, retardation, vesiculation of embryos and shell abnormality occurred (Text-fig. 2A).

Effect on 60-hr.-old embryos

A 1-hr, treatment of this stage did not seem to produce any effect. Continuoustreatment caused retardation of development of 120 to 168 hr. compared withthe control embryos, and also vesiculation and shell abnormalities. In somedevelopment was first retarded and then ceased.

Effect on 72-hr.-old embryos

These showed vesiculation in the head region, the foot or the visceral hump, andshell abnormalities (Text-fig. 2B & C), retardation or a complete block in develop-ment.


A curious observation in the azide experiments involving 60- and 72-hr.-oldembryos needs to be mentioned here. Though many developed normally andonly showed retardation in development, such embryos like those which showedshell abnormalities or vesiculation, failed to hatch from their capsules. However,

TEXT-FIG. 2A: Azide (0-001 M) treated embryo beginning at 48-hr, old stage. The absence ofshell to be noted.

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B: 72-hr, old embryo treated with azide (0-001M) continuously. The large vesiculation(ves.) to be noted.

if they were freed from their capsules they moved about like any normal embryo.In one batch the experimental embryos were under observation for 20 days afterthe corresponding control embryos had hatched out. These embryos were inazide for 77 hr. only. What prevents apparently normal embryos, let alone abnor-mal ones, from hatching is a question which we are unable to answer.

Carbohydrate metabolism in Planorbis exustus 21

C: 60-hr, old embryo under continuous treatment with azide at 0-001M. The lobe foot(ft.) and the reduced shell (Sh.) to be noted.

Series B experiments {sodium azide 0 • 002 M)

The results of the experiments in this series were similar to those of Series A.The cleavage-stage embryos developed normally till gastrulation began and thenceased to develop. When gastrulating embryos were treated the process ofgastrulation stopped almost immediately. Hydropia developed as usual, but inmany cases the embryos degenerated before hydropia became visible. Sixty-hour-old embryos showed delay in development which ultimately came to astandstill. The 72-hr.-old embryos when placed in azide ceased to develop anyfurther.

Series C experiments (sodium azide 0-003 M)

The results of these experiments differed from those of the two earlier series.It was observed that the experimental embryos showed delay in cleavage ranging

. from 10 to 35 min. The development then proceeded apparently normally up togastrulation and then stopped and the embryos became hydropic. The 24-hr.-oldembryos stopped gastrulating and became hydropic. In the 48-hr.-old ones, also,the development was arrested but the embryos degenerated before hydropiabecame visible. The older stages (60 and 72 hr.-old) underwent a complete blockof development.

Series D experiments (iodoacetamide)

The IAA experiments were restricted to a few embryos (135 with 87 controls)since they were required to serve a limited purpose. With high concentration ofIAA (0-00 1M) cleavage-stage embryos showed arrest of development, slightenlargement and rounding off of the blastomeres. Older embryos also died as aresult of the treatment. The lower concentration (0-0005 M) again had similareffect on cleavage-stage embryos but older ones showed delay in development and,in a low percentage, vesiculation or developed normally. Even with the lesser con-centration of 0 • 00025 M, cleavage-stage embryos showed the same abnormalities


but older ones developed normally. The lowest concentration tried (0-000 1M)did not affect the cleavage-stage embryos. Unlike CAP, IAA did not produceexogastrulation, which may be because the concentration was not suitable; forexample, a concentration between 0 • 00025 and 0 • 0001 M was not tried. A detailedstudy was not undertaken because we merely wanted to see if any similarityexisted between the effect of CAP and IAA. In fact, arrest of or delay in develop-ment, rounding off of the blastomeres and their enlargement, are produced byboth the substances.

DISCUSSION

The interference in the development of P. exustus by sodium azide resulted inretardation in development and certain shell abnormalities. Hall & Moog (1948)studied the growth rate in some amphibian embryos in the presence of azide atdifferent concentrations and showed that the rate of growth gradually declined tozero; they believed that this was a result of a progressive decrease in certain fac-tors affected by azide. We presume this is the case in the present experimentalembryos, too, in which we have observed everything from slight retardation indevelopment to its complete cessation.

Raven (1958) refers to the work of Mancuso who found that azide treatment ofearly cleavage embryos of Physa produced exogastrulae and of Attardo who ob-served similar exogastrulation in Bithynia. However, we did not find any effect oncleavage or exogastrulation in embryos ofPlanorbis which stopped developmentduring the process of gastrulation.

Brachet (1950) stated that azide is an inhibitor of cytochrome oxidase. Cyto-chemical study (see Raven, 1958) showed that shell glands of Aplysia, Bithynia orshells of Physa and Limnaea gave positive cytochrome oxidase reactions. Infact, treatment with azide or potassium cyanide causes various shell abnormalities(Raven, 1958). These observations might well explain the irregularities in theshell formation noticed in our experiments.

Accumulation of fluid causing hydropia and vesiculation was of very commonoccurrence in the azide treated embryos. The abnormal water distributioncaused in CAP-treated P. exustus has earlier been thought to be due to analteration in the -SH and -SS- balance. Lithium, also, was found to cause adisturbance of the water balance. Raven (1952) suggests that this might be dueto a general effect on the protoplasmic colloids or a specific inhibition of thelarval kidney. Geilenkirchen & Nijenhuis (1959) believe that 'impairment ofmorphogenetic potencies' of the endoderm inevitably entails hydropia. Accord-ing to Hess (see Geilenkirchen & Nijenhuis, 1959) if there is no normal intimatecontact between the endoderm and the ectoderm, vesiculation and hydropiaoccur. This is quite understandable because we find that accumulation of fluidbegins only after gastrulation has been blocked. As already stated in later stagesalso accumulation of fluid took place resulting in vesiculation of embryos. A


large number of such abnormal embryos were produced by azide; a few cases werealso found among the IAA-treated embryos. It would thus appear that thisabnormal water distribution is an unspecific reaction.

The metabolic phases in development

Sodium azide is said to inhibit certain processes of synthesis and assimila-tions (Clifton, 1946; Spiegelman, 1946). It is found to produce effects similar tothose produced by dinitrophenol which is said to effect an uncoupling of theoxidations and harnessing by phosphorylation the energy thus liberated (Loomis& Lipmann, 1948). Hall & Moog (1948), who studied its effect on certain am-phibian species, were of the opinion that their findings could be explained on thebasis that azide prevents phosphorylations which supply energy for the develop-mental processes and probably that the succinate oxidizing system is affected.

We are, therefore, inclined to suggest that the pre-gastrulation and post-gastrulation phases have different energy producing systems. In the former ametabolism which involves protein or fat and not a carbohydrate probably domi-nates. Thus the succession of energy sources seems to be different for the speciesstudied. This seems reasonable when we consider the work of Buglia, reported byRaven (1958), who found that the respiratory quotient (R.Q.) during the cleavageof Aplysia was 0 • 8-0 -85. Mayerhof (see Raven, 1958) stated that fat is the prin-cipal fuel. However, Baldwin (1935) found an R.Q. of 1 -05 and suggested thatcarbohydrate metabolism is dominant throughout the development of Limnaeastagnalis. Another group of animals which apparently does not conform to thenormal sequence of energy succession is the amphibia.

We shall now discuss the possible interference by IAA in the pre-gastrulationstage of development. It would be recalled at the outset that Mulherkar & Sherbet(1963) found that integrity of the -SH groups was of great importance in themorphogenesis of P. exustus. They also found that the phase of development fromthe 2nd cleavage to the 4th cleavage seemed to be CAP-susceptible and the periodbetween the 3rd cleavage and the 3rd cleavage-flattened stages seemed to be criticalfrom the point of view of production of exogastrulae. The CAP-susceptible phase,in general, coincided with the process of ooplasmic segregation taking place in thecleaving eggs and the critical phase in particular coincided with the establishmentof the -SH rich ectoplasm. The rounding off of the blastomeres and their enlarge-ment observed in the IAA-treated embryos could also be explained on the basisthat the -SH groups present in the cortical region of the eggs are acted upon by thechemical.

Let us now consider the question of arrest of development caused by IAA. Twoexplanations are possible. Rapkine & Brachet (1951) have observed that IAArapidly blocks development of certain amphibian embryos primarily by itsaction on the spindle. Mazia (1954,1955,1958) has shown that oxidation of the-SH groups into -SS- causes the folding of the fibrous proteins into globularproteins. CAP or IAA might then act in this way by inhibiting the formation of the


spindle. An alternative explanation is that these chemicals interfere with theenergy requirements of cleavage. The energy source (' energy reservoir') might bea phosphorylated compound. A further connection between carbohydratemetabolism and cleavage has also been indicated. Enzymes involved in bothanaerobic and aerobic carbohydrate metabolism contain -SH groups in theiractive centres. The -SH reactants affect the integrity of these active centres and in-hibit the metabolism. When the energy source falls below a certain level cleavagestops. Earlier in this paper it was suggested that during cleavage some metabolismother than that involving carbohydrates might be dominant and probably, on thewhole, the carbohydrate metabolism is eclipsed by the dominant metabolicsystem. It is conceivable that the energy requirements of cleavage might form asmall fraction of the total energy requirements of the cleaving egg. If it were so,only high concentrations of azide might be expected to affect cleavage. Theseexpectations, in fact, are fulfilled. The Series C experiments with 0 - 0 0 3 M azidehave indicated that cleavage-stage embryos are affected to some extent byazide and gastrulation movements are inhibited. Very high concentrations werenot tried because azide might then act by sheer toxicity rather than by interferingwith carbohydrate metabolism.

The vulnerability of gastrulation to azide has been striking. This clearlyindicates that the substrate oxidized to meet the energy requirements of thesemorphogenetic activities is a carbohydrate. It is known, for example, that' a highcarbohydrate metabolism is required for the morphogenetic movements ofgastrulation'(Brachet, 1960).

In the post-gastrulation phase, azide seems to dissociate growth metabolismfrom the metabolism of maintenance. The 48- and 60-hr.-old embryos stoppeddevelopment or showed delay in development, and the 72-hr .-old embryos also be-haved in the same manner. In Series B and C experiments, a complete cessation ofdevelopment was observed in all 48-hr.-old, most of the 60-hr.-old and all the 72-hr.-old embryos. Thus carbohydrates seem to be essential for growth metabolism.

The main conclusions could be summarized as follows: In the development ofP. exustus, a metabolism involving other substrates than a carbohydrate isdominant in the pre-gastrulation phase. The energy requirements of cleavage,which conceivably form a small part of the total requirements of the embryo, aremet by carbohydrate metabolism. The importance of carbohydrate metabolismfor morphogenetic activities of gastrulation is evidenced by the vulnerability ofthese stages to azide. The carbohydrate metabolism is also important for theprocesses of differentiation and growth.

SUMMARY

1. The metabolism ofPlanorbis exustus has been studied using sodium azideand iodoacetamide (IAA) at different concentrations.

2. Azide (0 • 001,0 • 002 and 0 • 003 M) treated cleavage-stage embryos developed


normally till they reached gastrulation when their development was arrested.Gastrulae stopped all morphogenetic activities. This was generally followedby hydropia. No exogastrulation was caused. The 48-, 60- and 72-hr.-oldembryos showed delay in development, vesiculation and shell abnormalities orunderwent a block in development.

3. Treatment with IAA caused arrest of development, rounding off andslight enlargement of the blastomeres.

4. The development of this molluscan species seems to be divisible into pre-gastrulation and post-gastrulation (including gastrulation) phases. In the pre-gastrulation phase, a metabolism involving other substrates than a carbohydrateis dominant. The energy requirement of cleavage, which conceivably forms asmall fraction of the total requirement of the embryo, is met by carbohydratemetabolism. The importance of the latter for morphogenetic activities is evirdenced by the vulnerability of these stages to azide. The carbohydrate metabolismis also important for the processes of differentiation and growth.

RESUME

Etude du metabolisme des hydrocarbones chez Planorbis exustus1. On a etudie le metabolisme de Planorbis exustus a l'aide de nitrure de

sodium et d'iodacetamide (IAA) a differentes concentrations.2., Les embryons en cours de segmentation traites au nitrure (0,001, 0,002 et

0,003 M) se sont developpes normalement jusqu'a ce qu'ils atteignent la gastrula-tion, stade ou le developpement s'est arrete. Les gastrulas ont cesse toute ac-tivite morphogenetique. Ceci a ete generalement suivi d'hydropisie. II n'y a paseu d'exogastrulation. Les embryons de 48, 60 et 72 heures ont montre un retardde developpement, une vesiculisation et des anomalies coquillieres, ou ont subiun blocage du developpement.

3. Le traitement a 1'IAA a proveque un arret du developpement, un arron-dissement et une legere dilation des blastomeres.

4. Le developpement de cette espece de Mollusques semble etre divisible endeux phases, pregastrulation et post-gastrulation (y compris la gastrulation).Dans la phase de pregastrulation domine un metabolisme impliquant d'autressubstrats qu'un hydrocarbone. Les besoins en energie de la segmentation, quiconstituent sans doute une petite fraction des besoins totaux d l'embryon, sontcouverts par le metabolisme des hydrocarbones. L'importance de ces dernierspour les activites morphogenetiques est mise en evidence par la vulnerabilite deces stades au nitrure de sodium. Le metabolisme hydrocarbone est egalementimportant pour les processus de differentiation et de croissance.

ACKNOWLEDGEMENTS

We are deeply indebted to Professor C. H. Waddington, F.R.S., for reading the paper andmaking suggestions for its improvement. We thank Professor D. R. Newth for helping us toprepare it and Dr Leela Mulherkar for providing facilities of work in the Department of


Zoology, University of Poona. One of us (G.V.S.) is grateful to the Government of India forthe award of a National Research Fellowship and the other (M. S. L.) wishes to express hergratitude to the University Grants Commission of India for a Junior Research Fellowship.

REFERENCES

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BRACHET, J. (1950). Chemical Embryology. New York: Interscience Publishers, Inc.BRACHET, J. (1960). The Biochemistry of Development. London: Pergamon Press.CLIFTON, C. E. (1946). Microbial association. Adv. Enzymol. 6, 267-305.GEILENKIRCHEN, W. L. M. & NIJENHUIS, E. D. (1959). The influence of lithium chloride on

the development of embryos of Limnaea stagnalis treated during and after the gastrulastage. Konikl. Ned. Akad. Weten. 62, 214-24.

HALL, T. S. & MOOG, F. (1947). Effect of sodium azide on the rate of amphibian development(abstract). Biol. Bull. 93, 213.

HALL, T. S. & MOOG, F. (1948). Sodium azide as an inhibitor of amphibian development./ . exp. Zool. 109, 339-54.

LOOMIS, F. & LIPMANN, F. (1948). Reversible inhibition of the coupling between phosphory-lation and oxidation. / . biol. Chem. 173, 806-8.

LOVTRUP, S. (1959«). On the causes of variation in the respiratory curves during amphibianembryogenesis. / . exp. Zool. 140, 231-46.

L0VTRUP, S. (19596). Utilization of energy sources during amphibian embryogenesis at lowtemperatures. / . exp. Zool. 140, 383-94.

MAZIA, D. (1954). The particulate organization of chromosome. Proc.nat. Acad. Sci., Wash.40, 521-7.

MAZIA, D. (1955). The organization of the mitotic apparatus. Symp. Soc. exp. Biol. 9,335-57.

MAZIA, D. (1958). SH compounds in mitosis. 1. The action of mercaptoethanol on the eggsof the sand dollar Dendraster excentricus. Exp. Cell Res. 14,486-94.

MULHERKAR, L. & SHERBET, G. V. (1963). The effect of chloroacetophenone on the develop-ment of Planorbis {Indoplanorbis) exustus (Deshayes). / . Embryo!, exp. Morph. 11,53-64.

NEEDHAM, J. (1950). Biochemistry and Morphogenesis. Cambridge: The University Press.RAPKINE, L. & BRACHET, J. (1951). Recherches sur le role des groupes sulfhydriles dans la

morphogenese. 1. Action des inhibiteurs des groupes -SH sur l'oeuf entier et sur desexplantat dorsaux et ventraux chez les Amphibiens. Implantation des proteines sulf-hydrilees. Bull. Soc. Chim. biol, Paris, 33,427-38.

RAVEN, CHR. P. (1952). Morphogenesis in Limnaea stagnalis and its disturbance by lithium./. exp. Zool. 121, 1-77.

RAVEN, CHR. P. (1958). Morphogenesis: The Analysis of Molluscan Development. London:Pergamon Press.

SPIEGELMAN, S. (1946). Nuclear and cytoplasmic factors controlling enzymatic constitution.ColdSpr. Harb. Symp. 11, 256-77.

{Manuscript received 28th June 1963)

a study of carbohydrate metabolism in planorbis …2015/12/01 · embryol. exp. morph., vol. 12,...

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