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Stimulation of RNA and protein synthesis in silkmoth pupal wing tissue by ecdysone in vitro
GENERAL
AND
COMPARATIVE
ENDOCRINOLOGY
Stimulation
of Pupal
Department
RNA
16, 369-374 (1971)
and
Wing
Tissue
S. SILVER
WYATT
of
Biology,
Yale
Protein
Synthesis
by Ecdysone AND
University,
in Silkmoth
in Vitro
G. R. WYATT New
Haven,
Connecticut
06620
Received June 22, 1970 A mince of wing tissue from pupae of the silkmoth, Hyalophora cecropiu, in diapause was incubated in a physiological medium, and effects of hormones on biosynthesis were examined. Both RNA and protein synthesis, measured by incorporation of precursors for 1-2 hours, were significantly elevated after 20 hours with P-ecdysone. In stimulating RNA synthesis, P-ecdysone was effective at l-20 pg/ml and less so at 0.2 pg/ml. At 5 pg/ml, ,&ecdysone gave, on the average, 65% stimulation of RNA synthesis and was significantly more active than cu-ecdysone (30% stimulation), while 22-isoecdysone had no effect.
In the study of hormone action, in order to permit control of conditions and avoid interactions between tissues, it is important to have experimental systems in which isolated tissue responds to hormone added in vitro. It was with this object that media and conditions for incubation of silkmoth fat body (Stevenson and Wyatt, 1962) and epidermal tissue (Reddy and Wyatt, 1967) were developed in this laboratory. New synthesis of nucleic acids and proteins has a central role in the growth-promoting action of steroid hormones (Tata, 1966)) including ecdysone (Karlson and Sekeris, 1966; Neufeld et al., 1968; Gore11 and Gilbert, 1969). Insect epidermis is an established target tissue of ecdysone, and the wing epidermis of silkmoth pupae in diapause exhibits enhanced RNA and protein synthesis within 6-24 hours after injection of this hormone (Wyatt, 1967, 1970). We have therefore now examined the effect of ecdysonel on these processes in Cecropia pupal wing tissue in vitro, and have found a significant stimulation. ‘In this paper, ecdysone is used in a collective sense to denote steroids with insect molting hormone activity. a-ecdysone is 2,8,3,~3,14n,22R.25pentahydroxy-A’-5P-cholesten-6-one (often called simply ecdysone), and P-ecdysone is 20-OH+ecdysone (=ecdysterone) (cf. Wyatt, 19711. 369
MATERIALS
AND
METHODS
Materials The saturniid silkmoth, Hyalophora cecropia, was reared out of doors on wild cherry foliage, or in the laboratory (25”C, 60% RH) on the artificial diet of Riddiford (lW8). Similar results were obtained with animals from either source. All experiments were performed with pupae placed in permanent diapause by removal of the brain (Williams, 1946)) and their low metabolism was confirmed by manometri: measurement of respiration (Schneiderman and Williams, 1953). Uridine-2-V (32 mCi/mmole) and cytidine5-“H (26 Ci/mmole) were purchased from New England Nuclear Corporation. Boston, Massachusetts, and leucine-4,5-“H (45 Ci/mmole) from Schwarz BioResearch, New York. Crystalline synthetic a-ecdysone was given by Dr. A. D. Cross, Syntex Research, Palo Slto, California, and 22-iso-a-ecdysone by Dr. J. B. Siddall, Zoecon Corporation, Palo Alto, California. P-Ecdysone (of plant origin) was purchased from Mann Research Laboratories, New York.
Preparation and Incubation of Tissue Incubation medium was prepared according to Reddy and Wyatt (19671. The inclusion of n-leucine (1 mM) when incorporation of radioactive leucine was to be measured was important in order to minimize variation in dilution of the isotope by amino acid from the hemolymph contained in the wing tissue. When uridine or
370
\VY.-\TT
AND
cptidine incorporation was meaaurecl, 08.025 mill uridine or 0.05 mllf cytidim. rcspcc~tivc~ly. wns included in the medium. Sterile technique was as previously describccl (Reddy and Wyatt, 1967), except that for t,hc external disinfection of pupae Clorox was replaced by 70% ethanol. At. the conclusion of (very experiment, samples of the medium were streaked on nutrient agar, and in the rare CUSP of microbial growth, results were discarded. The procedure was as described previous1.v with the following exceptions. Some hcmolymph was collected from each pupa onto a few crystals of phenylthiourea, and was then centrifuged at 2500 rpm for 12 min to provide plasma. Wings collected in potassium-Ringer solution were KC1 154 mM, NaCl 5.6 mM, CaCl? 2.3 mM, brought to pH 7.0 with about 0.1 mM NaHCOB) containing a little phenylthiourea. After being minced with scissors. the tissue was shaken gently in incubation medium at 0°C for 15 min in order to rinse out hemolymph. By centrifuging at 200 g (1000 rpm) for 2 min, the tissue was collected into a pellet. This was drained, slid onto a petri plate, and then divided with spatulas into equalappearing portions which were distributed into the incubation tubes. We a!so tried distribution of the minced wing tissue hy pipetting it from but this achieved no greater unisuspension, formity. In most experiments, wings from 8 pupae. minced and rinsed in 8 ml of medium, provided the tissue for 8 incubation tubes, each containing 0.5 ml of medium.
Preparation Procedure
for Counting A, used
in the earlier experiments for measuring incorporation of both uridine-‘“C and leucine-‘H, was slightly modified from that of Reddy and Wyatt (1967) according to the recommendations of Munro and Fleck (1966). Incorporation was stopped by the addition of 2 ml of cold 0.5 N HCIO1, the tissue was ground in a glass homogenizer, and the residue was collected by centrifuging and washed 3X with cold 0.4N HClO+ then once each with ethanol (95% containing 2% sodium acetate), ethanol (95%0), ethanol-ether (3: I), and ether. Total nucleic acids were extracted with hot HClO, and the extract and residue (protein) were prepared for counting as previously described. These were counted in a Packard scintillation counter (Model 3003) and corrected for quenching by use of the instrument’s external standard. Results are expressed as dpm/ pmole total nucleic acid (RNA + DNA). Procedure B, used in later experiments for incorporation into RNA, proved to be less tedious and to give more reproducible results. It was based
WT.1I-I
on that of Howclls :md Wyatt (1969). \vllic,ll. applied to this tissut~. gave c,fficicmt cstraction of RNA contaminated wit.11 ahout 2% of DSA and 5% of protein. The separation f;om Di’i\;A pPrmil,ted the use of cytidine-“H as :L precursor. which is incorporated more efIi&ntly than uridine in silkmoth wing tissue (Lucas, 19681, probably because of the pool of uridinr nucleotides. After incubation, the tubes were chilled in ice and the tissue was pelleted by centrifugation and drained. To each tube was added 2 ml sodium acetate buffer (0.05M. pH 5.0) and 0.2 ml 5% sodium dodecyl sulfate, the tissue was homogenized briefly in a Teflon-glass tissue grinder, then 2 ml 80% phenol was added and the mixture was homogenized well. Each tube was heated to 55°C for 2 min and mixed thoroughly again. After cooling and centrifugation (10 min at 10,000 rpm in the Servall RC-2) , the upper layer was drawn off. This was retreated with 2 ml fresh 80% phenol and again centrifuged and drawn off. KC1 (0.2 ml l.OM) and ethanol (5 ml 95%) were added and the tubes were left at -20” overnight. The fine precipitate of RNA was collected by centrifuging (IO min at 15,C00 rpm), drained, washed twice with 2 ml of 95% ethanol with care to rinse and drain the walls of the tube, and finally dissolved in 0.5 ml acetate buffer (as above). Portions of this solution were diluted for estimation of RNA by absorbance at 260 nm [mE(P) = 8.01. Samples of 0.1 ml for measurement of radioactivity were delivered onto 25 mm circles of Whatman 3MM filter paper which were then washed with 0.25 N HClO, (on ice), 95% ethanol (twice), ethanolether (3: l), and ether, and dried before counting in 5 ml of toluene phosphor. Since the counting efficiency was unknown but presumably constant, results from this procedure are expressed as counts per minute per micromole of RNA. RESULTS
In Fig. 1 is shown incorporation into RNA and protein in wing tissue from pupae in diapause and from animals induced to develop by injection of ecdysone 20 hours previously. The results illustrate the elevated rates of both processesconsequent to ecdysone action in vivo, and show that 1 hour is a suitable incorporation time for detecting differences. This time was used routinely in later experiments. Effects of p-ecdysone on RNA and protein synthesis after addition to tissue in vitro are shown in Table 1. After 10 hours with the hormone, both processes appear to be somewhat elevated, but, from the
ECDYSONE
EFFECT
OF &ECDYSONE
ON INCORPORATION
Uridine-‘4C Duration of treatment (Hours)
No. of Expts.
10 20
2 3
AND
incorporation
SYNTHESIS
IN
MOTH
TABLE 1 OF URIDINE
AND LEUCINE
(dpmlpmole
371
WINGS
Leucine-ZH
INTO WING incorporation
NA)
Control (0
+&ECdysone (W
E c
3212 4685
3925 8701
1.59 2.88
TISSUE
in
Vitro”
(dpm/pmole
NA)
5~ SE&l* k 0.36 f 0.16
p”
Control ((-2
+j%Ecdysone (W
NS
11,892 12,290
15,008 18,883
E c If: SEM* 1.26 1.57
+ 0.10 _+ 0.01
pc R‘S <0.05
a Tissue from pupae in diapause was incubated with or without fl-ecdysone (5 fig/ml) for 10 or 20 hours, during the last hour of which the isotopic compounds, as in Fig. I, were added for incorporation. Samples were prepared for counting by procedure A. Results shown are means of values from the separate experiments. 6 The values given for E/C are the means of the individual values for this ratio, and are therefore not identical with the ratios of the mean values of E and C given in the table. c P is probability that E/C = 1.00, by t test.
limited data, the difference from controls is not significant. At 20 hours, the effect is greater and statistically significant. The experiments reported in Table 2 tested the effects of 20 hours exposure to different levels of ,&ecdysone on incorporation into RNA. Hemolymph plasma was included in the medium, as discussed below.
From these data, the effects of 1, 5, or 20 pg/ml P-ecdysone are not distinguishable, whereas that of 0.2 yg/ml is distinctly less. In Table 3 are presented further results on the effects of /3-ecdysone, a-ecdysone, and 22-isoecdysone, tested at 5 pg/ml for 20 hours, as well as some variations in the medium. Comparison A shows that the presence of 20% hemolymph (from diapause) more than doubles the rate of cytidine incorporation. Earlier experiments
EFFECT
r-Ecdysone Gghl) Incorporatton
time
(hoursF
FIG. 1. Incorporation of uridine and leucine in vitro into wing tissue from diapause and developing cecropia. Pupae in diapause (3 months old) were injected with 20 rg c-ecdysone then kept 19 hours at 25”; controls received no injection. Wing tissue was excised and samples were incubated in 0.5 ml medium with uridineJ4C (0.5 &i) and leucin@H (5.0 &i) for 1 or 2 hours. Samples were prepared by procedure A for counting. Each point is the average from duplicate tubes in a single experiment. -•--, uridine incorporation, diapause; - 0-, uridine after ecdysone; --A-, leucine, diapause; --A-, leucine after ecdysone.
0 0.2 1 5 20
TABLE 2 OF DIFFERENT LEVELS OF ,%ECDYSONE ON RNA SYNTHESIS’ Specific
activity (wdmole)
843.5 8640 13000 11510 12090
of RNA
(9020, 7850) (8135, 9145) (14200, 11800) (12025, 10990) (11075, 13110)
a The medium consisted of 0.4 ml of synthetic medium + 0.1 ml of diapause pupal hemolymph plasma. p-Ecdysone dissolved in water was added as indicated. Tissue from pupae in diapause was added and incubated at 25” for 19 hours, then cytidineJH (2.5 &i) was added and incubation was continued for 1 hour. Samples were prepared for counting by procedure B. The means and individual results from two experiments are shown.
Modifications Comparison A B c D
Conk01
- Hemolymph +Ethanol
-
+p-Ecdysone +@-Ecdysone
+Ethanol -
in ethanol in ethanol
+Ethanol
+22-Isoecdysone ethanol
in
Exptl. (E)
E c + SEW
Pb
4985 6025
2280 6063
0.41 0.99
zk 0.01 k 0.14
7 3
4914 5773 ~5172
8231 11503
1.56 1.93
z!z 0.09 * 0.19
9313
1.67
& 0.10
7913 5122 __ 6518
1.41 1.15
+ 0.14 k 0.14
6
5765 -.4359 5062
1.28
f 0.10
<0.05
2
6375
6667
1.05
k 0.05
NS
iii
+a-Ecdysone (combined)
E+F G
+a-Ecdysone +a-Ecdysone
-Control CC)
4 2 in ethanol
+p-Ecdysone (combined)
C+D E F
No. of Expts.
Experimental
+Ethanol
-
to medium
Specific act,ivit? of RKA (cpm/ pmole)
3 3
a The standard medium and conaitions were as described in Table 2. Steroids were used at 5 pg/ml. Ethanol, when used as a solventj or in controls, was 25 ~1 of 10%;. The data from a series of 12 experiments (each comparing four different conditions) are assembled as comparisons of the mean result for each experimental condition with that for the corresponding control. a See footnotes to Table 1.
by procedure A (data not shown) indicated that hemolymph from animals in diapause or development was about equally effective. Its effect was on preservation of celnot on the incorporation lular activity, process, for hemolymph added at the same time as the isotope, after 19 hours incubation of tissue without it, did not stimulate. In all later experiments, hemolymph was included in the medium. Hormonal effect’s were obtained both without added hemolymph (Table 1) and with it (Tables 2 and 3). Since some hemolymph was always carried in the wing lacunae, however, we cannot tell whether the tissue might respond to ecdysone in its complete absence. Comparison B shows that ethanol, in the small amount used as a solvent, had no effect. The stimulatory effect of ,&ecdysone on RNA synthesis, shown in comparisons C and D, is highly significant. In individual experiments, E/C ranged from 1.19 to 2.17. The apparently greater effect of P-ecdysone in ethanol (D) than in water (C) is not
statistically significant (P > 0.1). The results with #cY-ecdysone are shown in comparisons E and F ; in some experiments the ethanol control was omitted since no difference had been found from the water control. The effect of a-ecdysone is less than that of P-ecdysone at the same concentration, and in some experiments no effect was observed (E/C ranged from 0.98 to 1.68). When the 6 experiments with cu-ecdysone are combined (E + F), however, the stimulation is st’atistically significant. Also, comparison of the combined data for a-ecdysone (E + F) with those for /?-ecdysone (C + D) shows that the difference between the effects of the two hormones is significant (2’ < 0.02). 22-Isoecdysone (comparison G) was inactive. DISCUSSION
Since pure ecdysones have become available, cytological and developmental effects of these hormones upon several insect tissues in vitro have been described (review: Wyatt, 1971). Stimulation of RNA syn-
ECDYSOKE
AND
SYNTHESIS
thesis in isolated insect cell nuclei by added ecdysone has also been claimed (Sekeris et al., 1965; Congote et al., 1969). The only biochemical response of an intact insect tissue to ecdysone in vitro yet reported is that of DNA synthesis in imaginal discs of the wax moth (Galleria mellonella), demonstrated by autoradiography (Oberlander, 1969). In the present paper we add quantitative demonstration of stimulation of RNA and protein synthesis by ecdysone added to isolated intact insect tissue. After incubating wing tissue from Cecropia in diapauee for 20 hours with p-ecdysone at 5 pg/ml, we find rates of RNA and protein synthesis elevated, on the average, about 65% above controls, and there was some positive effect in every experiment. With #a-ecdysone, on the other hand, the stimulation was less (average 30% above controls) and variable, so that in some experiments none was seen. The apparent ineffectiveness of a-ecdysone (2 ,pg/ml) in an experiment reported previously (Retldy and Wyatt, 1967) is therefore not inconsistent wit,h t,he present data. The specificity of the effect is further shown by the inactivity of 22-isoecdyeone, an analog which is inert in other ecdysoneresponsive systems (Furlenmeier et al.. 1967; .Judy, 1969; Marks and Leopold, 1970). The greater effect of /3- than cy-ecdysone is interesting in relation to the ouestion of their respective biological roles. Although a-ecdysone was the first to be isolated in pure form from an insect source, recent work indicates that insects generally contain more fi-e?dysone (review: Wyatt, 1971). Also, rapid conversion of cr- to /3ecdysone (20-hydroxylation) in insects has been demonstrated (King and Siddall, 1969; Moriyama et al., 1970). It is thus quite likely that P-ecdysone is the more generally active form of the hormone. On the other hand, DNA synthesis in Gal&a wing discs in vitro is reported to respond to a-ecdysone but not to ,&ecdyeone (Oberlander, 1969). Comparison of dose and time relationships in the action of the two ecdvsones in vitro should clarifv their roles. The increase in hiosynt,hesis after in-
IN
MOTH
WINGS
373
cubation of tissue with p-ecdysone for 20 hours is comparable to that found in tissue from animals in which the hormone had been injected 20 hours earlier (Fig. 1). Both, however, are much smaller than the elevation found by measurement in vivo after a similar time of ecdysone action, when incorporation int’o both RNA and protein may be elevated as much as IO-fold (Wyatt, 1970). The smaller apparent hormonal stimulation in vitro may be due to increased activity in the control tissue as a result of stripping from the pupal cuticle, as has been pointed out previously (Reddy and Wyatt, 1967). The stimulatory effect of ecdysone on cecropia wing tissue encourages us to continue the biochemical study of hormonal action on insect tissues in vitro. ACKNOWLEDGMENTS We are grateful J. B. Siddall for gifts work was supported Foundation and the U. S. Public Health
to
Dr. 9. D. Cross and Dr. of synthetic hormones. This by grants from the Whitehall National Institutes of Health, Service (HD-02176).
REFERENCES L. F., SEKERIS, C. E., .~ND KARLSON, P. (1969). On the mechanism of hormone action. XIII. Stimulating effects of ecdysone, juvenile hormone, and ions on RNA synthesis in fat body cell nuclei from Culliphora erythrocephaln isolated by a filtration technique. Exp. Cell Res. 56, 338-346. FURLENMEIER, A., F~~RsT. A.. 1J~~~~~~4~~. -4., WPILDVOGEL: G., HOCKS. P.. KERB, U.. AND WEICHERT, R. (1967). Zur Synthese des Ecdysons. IX. Mitteilung iiber Insektenhormone. Hefv. Chim. Actn 50, 2387-2396. GORELL, T. A.. AND GILBZRT. I,. I. (1969). Stimulation of protein and R,NA synthesis in the crapfish hepatopancreas by crustecdysone. Gen. Comp. Endocrinol. 13, 308-310. HOWELLS, A. M., AND WYATT, G. R. (1969). The apparent template activity of RNA from developing wings of the cecropia silkmoth. Biochim. Biophys. Acta 174, 86-98. KARLSON. P., AND SEKERIS. C. E. (1966). Ecdysone. an insect steroid hormonr. and its mode of action. Recent Progr. Helm. Res. 22, 473-502. KING, D. S.. AND SIDDALL, J. B. (1969). Conversion of cr-ecdysone to P-ecdysonc by crustaceans and insects, Nntxre (London) 221, 956956. CONGOTE,
374
\VT.-\TT
ANI)
JUUY. K. J. (1969). Cellular resl,onsc: to c~c++ terone itl uitro. Science 165, 137441375. LUCAS. K. U. (1968). DSA-RNA hybridization studies on the developing wings of thcx c,ccropia silkmoth. Ph.D. dirsertation. Yale I’nitersity. New Haven. Connecticut. MARKS, E. P., AND LEOPOLD, R. A. (1970). Cockroach leg regeneration: effects of ec*dyst,erone in vitro. Science 167, 61-62. MORIYAMA, H.. NAKANISHI, K.. KING, D. S.. OKAUCHI, T., SIDDALL, J. B.. AND HAFFERL, W. (1970). On the origin and metabolic fate of a-ecdysone in insects. Gen. Comp. Endocrinol., 15, 80. MUNRO. H. N., AND FLECK, A. (1966). The determination of nucleic acids. Metk. Biochem. Anal. 14, 113-176. NEUFELD, G. J., THOMSON, J. A., AND HORN, D. S. H. (1968). Short-term effects of crustecdysone (20-hydroxyecdysone) on protein and RNA synthesis in third-instar larvae of Calliphora. J. Insect Physiol. 14, 789-802. OBERLANDER, H. (1969). Ecdysone and DN.4 synthesis in cultured wing disks of the was moth, Galleria mellonellx J. Insect Physiol. 15, 18031806. REDDY, S. R. R.. AND WYATT, G. R. (1967). Incorporation of uridine and leucine in vitro by cecropia silkmoth wing epidermis during diapause and development. J. Insect Physiol. 13, 981-994. RIDDIFORD, L. M. (1968). An artificial diet for
\VY:\TT
H. I-\.. .\~a RILLIIMS. C. IQ. (1953). The physiology of insf,ct di:cl~xu~i’~. VII. The resl+:itory met.aboli+m of the I.ecropia silkworm during diapauac and develol~ment. Biol. Bull. 105, 320-334. SEKERIS. C. l3., DUKES. P. P., ASD S;(IHMII). W. (1965). Wirkung von Ecdyson auf Flpidcrmiszellkerne von Cnlliphom I,ar\-cn i)t ~~ih. IIoppeSeyler’s Z. Phusiol. Chenz. 341, 152-154. STEVENSON, E.. AND wy.4~~. G. R. (1962). The metabolism of silkmoth tissues. Incorporation of leucine into protein. Arch. Biochenl. Biophys. 99, 65-71. TATA. J. R. (1966). Hormones and the synthesis and utilization of ribonucleic acids. Progr. N~cl. Acid RPS. Mol. Biol. 5, 191-250. WILLIAMS. C. M. (1946). Physiology of insect diapause: the role of the brain in the production and termination of pupal dormanq in the giant silkworm. P. cecropin. Biol. Bull. 90, 234243. WYATT, G. R. (1967). Macromolecular hiosynthesis in insect metamorphosis. Inl. (‘ongr. Biochem., 7th, Tokyo, Abstr. 2. 389. WYATT. G. R. (1970). Nucleic acid and protein synthesis in hormone-induced development in silkmoth pupae. In preparation. WYATT. G. R. (1971). Insect hormones. In “Biochemical Actions of Hormones” (G. Litwack. ed.), Vol. 2. Academic Press, New York. (In press.) SVIISEII~ISHX~S.
AND
COMPARATIVE
ENDOCRINOLOGY
Stimulation
of Pupal
Department
RNA
16, 369-374 (1971)
and
Wing
Tissue
S. SILVER
WYATT
of
Biology,
Yale
Protein
Synthesis
by Ecdysone AND
University,
in Silkmoth
in Vitro
G. R. WYATT New
Haven,
Connecticut
06620
Received June 22, 1970 A mince of wing tissue from pupae of the silkmoth, Hyalophora cecropiu, in diapause was incubated in a physiological medium, and effects of hormones on biosynthesis were examined. Both RNA and protein synthesis, measured by incorporation of precursors for 1-2 hours, were significantly elevated after 20 hours with P-ecdysone. In stimulating RNA synthesis, P-ecdysone was effective at l-20 pg/ml and less so at 0.2 pg/ml. At 5 pg/ml, ,&ecdysone gave, on the average, 65% stimulation of RNA synthesis and was significantly more active than cu-ecdysone (30% stimulation), while 22-isoecdysone had no effect.
In the study of hormone action, in order to permit control of conditions and avoid interactions between tissues, it is important to have experimental systems in which isolated tissue responds to hormone added in vitro. It was with this object that media and conditions for incubation of silkmoth fat body (Stevenson and Wyatt, 1962) and epidermal tissue (Reddy and Wyatt, 1967) were developed in this laboratory. New synthesis of nucleic acids and proteins has a central role in the growth-promoting action of steroid hormones (Tata, 1966)) including ecdysone (Karlson and Sekeris, 1966; Neufeld et al., 1968; Gore11 and Gilbert, 1969). Insect epidermis is an established target tissue of ecdysone, and the wing epidermis of silkmoth pupae in diapause exhibits enhanced RNA and protein synthesis within 6-24 hours after injection of this hormone (Wyatt, 1967, 1970). We have therefore now examined the effect of ecdysonel on these processes in Cecropia pupal wing tissue in vitro, and have found a significant stimulation. ‘In this paper, ecdysone is used in a collective sense to denote steroids with insect molting hormone activity. a-ecdysone is 2,8,3,~3,14n,22R.25pentahydroxy-A’-5P-cholesten-6-one (often called simply ecdysone), and P-ecdysone is 20-OH+ecdysone (=ecdysterone) (cf. Wyatt, 19711. 369
MATERIALS
AND
METHODS
Materials The saturniid silkmoth, Hyalophora cecropia, was reared out of doors on wild cherry foliage, or in the laboratory (25”C, 60% RH) on the artificial diet of Riddiford (lW8). Similar results were obtained with animals from either source. All experiments were performed with pupae placed in permanent diapause by removal of the brain (Williams, 1946)) and their low metabolism was confirmed by manometri: measurement of respiration (Schneiderman and Williams, 1953). Uridine-2-V (32 mCi/mmole) and cytidine5-“H (26 Ci/mmole) were purchased from New England Nuclear Corporation. Boston, Massachusetts, and leucine-4,5-“H (45 Ci/mmole) from Schwarz BioResearch, New York. Crystalline synthetic a-ecdysone was given by Dr. A. D. Cross, Syntex Research, Palo Slto, California, and 22-iso-a-ecdysone by Dr. J. B. Siddall, Zoecon Corporation, Palo Alto, California. P-Ecdysone (of plant origin) was purchased from Mann Research Laboratories, New York.
Preparation and Incubation of Tissue Incubation medium was prepared according to Reddy and Wyatt (19671. The inclusion of n-leucine (1 mM) when incorporation of radioactive leucine was to be measured was important in order to minimize variation in dilution of the isotope by amino acid from the hemolymph contained in the wing tissue. When uridine or
370
\VY.-\TT
AND
cptidine incorporation was meaaurecl, 08.025 mill uridine or 0.05 mllf cytidim. rcspcc~tivc~ly. wns included in the medium. Sterile technique was as previously describccl (Reddy and Wyatt, 1967), except that for t,hc external disinfection of pupae Clorox was replaced by 70% ethanol. At. the conclusion of (very experiment, samples of the medium were streaked on nutrient agar, and in the rare CUSP of microbial growth, results were discarded. The procedure was as described previous1.v with the following exceptions. Some hcmolymph was collected from each pupa onto a few crystals of phenylthiourea, and was then centrifuged at 2500 rpm for 12 min to provide plasma. Wings collected in potassium-Ringer solution were KC1 154 mM, NaCl 5.6 mM, CaCl? 2.3 mM, brought to pH 7.0 with about 0.1 mM NaHCOB) containing a little phenylthiourea. After being minced with scissors. the tissue was shaken gently in incubation medium at 0°C for 15 min in order to rinse out hemolymph. By centrifuging at 200 g (1000 rpm) for 2 min, the tissue was collected into a pellet. This was drained, slid onto a petri plate, and then divided with spatulas into equalappearing portions which were distributed into the incubation tubes. We a!so tried distribution of the minced wing tissue hy pipetting it from but this achieved no greater unisuspension, formity. In most experiments, wings from 8 pupae. minced and rinsed in 8 ml of medium, provided the tissue for 8 incubation tubes, each containing 0.5 ml of medium.
Preparation Procedure
for Counting A, used
in the earlier experiments for measuring incorporation of both uridine-‘“C and leucine-‘H, was slightly modified from that of Reddy and Wyatt (1967) according to the recommendations of Munro and Fleck (1966). Incorporation was stopped by the addition of 2 ml of cold 0.5 N HCIO1, the tissue was ground in a glass homogenizer, and the residue was collected by centrifuging and washed 3X with cold 0.4N HClO+ then once each with ethanol (95% containing 2% sodium acetate), ethanol (95%0), ethanol-ether (3: I), and ether. Total nucleic acids were extracted with hot HClO, and the extract and residue (protein) were prepared for counting as previously described. These were counted in a Packard scintillation counter (Model 3003) and corrected for quenching by use of the instrument’s external standard. Results are expressed as dpm/ pmole total nucleic acid (RNA + DNA). Procedure B, used in later experiments for incorporation into RNA, proved to be less tedious and to give more reproducible results. It was based
WT.1I-I
on that of Howclls :md Wyatt (1969). \vllic,ll. applied to this tissut~. gave c,fficicmt cstraction of RNA contaminated wit.11 ahout 2% of DSA and 5% of protein. The separation f;om Di’i\;A pPrmil,ted the use of cytidine-“H as :L precursor. which is incorporated more efIi&ntly than uridine in silkmoth wing tissue (Lucas, 19681, probably because of the pool of uridinr nucleotides. After incubation, the tubes were chilled in ice and the tissue was pelleted by centrifugation and drained. To each tube was added 2 ml sodium acetate buffer (0.05M. pH 5.0) and 0.2 ml 5% sodium dodecyl sulfate, the tissue was homogenized briefly in a Teflon-glass tissue grinder, then 2 ml 80% phenol was added and the mixture was homogenized well. Each tube was heated to 55°C for 2 min and mixed thoroughly again. After cooling and centrifugation (10 min at 10,000 rpm in the Servall RC-2) , the upper layer was drawn off. This was retreated with 2 ml fresh 80% phenol and again centrifuged and drawn off. KC1 (0.2 ml l.OM) and ethanol (5 ml 95%) were added and the tubes were left at -20” overnight. The fine precipitate of RNA was collected by centrifuging (IO min at 15,C00 rpm), drained, washed twice with 2 ml of 95% ethanol with care to rinse and drain the walls of the tube, and finally dissolved in 0.5 ml acetate buffer (as above). Portions of this solution were diluted for estimation of RNA by absorbance at 260 nm [mE(P) = 8.01. Samples of 0.1 ml for measurement of radioactivity were delivered onto 25 mm circles of Whatman 3MM filter paper which were then washed with 0.25 N HClO, (on ice), 95% ethanol (twice), ethanolether (3: l), and ether, and dried before counting in 5 ml of toluene phosphor. Since the counting efficiency was unknown but presumably constant, results from this procedure are expressed as counts per minute per micromole of RNA. RESULTS
In Fig. 1 is shown incorporation into RNA and protein in wing tissue from pupae in diapause and from animals induced to develop by injection of ecdysone 20 hours previously. The results illustrate the elevated rates of both processesconsequent to ecdysone action in vivo, and show that 1 hour is a suitable incorporation time for detecting differences. This time was used routinely in later experiments. Effects of p-ecdysone on RNA and protein synthesis after addition to tissue in vitro are shown in Table 1. After 10 hours with the hormone, both processes appear to be somewhat elevated, but, from the
ECDYSONE
EFFECT
OF &ECDYSONE
ON INCORPORATION
Uridine-‘4C Duration of treatment (Hours)
No. of Expts.
10 20
2 3
AND
incorporation
SYNTHESIS
IN
MOTH
TABLE 1 OF URIDINE
AND LEUCINE
(dpmlpmole
371
WINGS
Leucine-ZH
INTO WING incorporation
NA)
Control (0
+&ECdysone (W
E c
3212 4685
3925 8701
1.59 2.88
TISSUE
in
Vitro”
(dpm/pmole
NA)
5~ SE&l* k 0.36 f 0.16
p”
Control ((-2
+j%Ecdysone (W
NS
11,892 12,290
15,008 18,883
E c If: SEM* 1.26 1.57
+ 0.10 _+ 0.01
pc R‘S <0.05
a Tissue from pupae in diapause was incubated with or without fl-ecdysone (5 fig/ml) for 10 or 20 hours, during the last hour of which the isotopic compounds, as in Fig. I, were added for incorporation. Samples were prepared for counting by procedure A. Results shown are means of values from the separate experiments. 6 The values given for E/C are the means of the individual values for this ratio, and are therefore not identical with the ratios of the mean values of E and C given in the table. c P is probability that E/C = 1.00, by t test.
limited data, the difference from controls is not significant. At 20 hours, the effect is greater and statistically significant. The experiments reported in Table 2 tested the effects of 20 hours exposure to different levels of ,&ecdysone on incorporation into RNA. Hemolymph plasma was included in the medium, as discussed below.
From these data, the effects of 1, 5, or 20 pg/ml P-ecdysone are not distinguishable, whereas that of 0.2 yg/ml is distinctly less. In Table 3 are presented further results on the effects of /3-ecdysone, a-ecdysone, and 22-isoecdysone, tested at 5 pg/ml for 20 hours, as well as some variations in the medium. Comparison A shows that the presence of 20% hemolymph (from diapause) more than doubles the rate of cytidine incorporation. Earlier experiments
EFFECT
r-Ecdysone Gghl) Incorporatton
time
(hoursF
FIG. 1. Incorporation of uridine and leucine in vitro into wing tissue from diapause and developing cecropia. Pupae in diapause (3 months old) were injected with 20 rg c-ecdysone then kept 19 hours at 25”; controls received no injection. Wing tissue was excised and samples were incubated in 0.5 ml medium with uridineJ4C (0.5 &i) and leucin@H (5.0 &i) for 1 or 2 hours. Samples were prepared by procedure A for counting. Each point is the average from duplicate tubes in a single experiment. -•--, uridine incorporation, diapause; - 0-, uridine after ecdysone; --A-, leucine, diapause; --A-, leucine after ecdysone.
0 0.2 1 5 20
TABLE 2 OF DIFFERENT LEVELS OF ,%ECDYSONE ON RNA SYNTHESIS’ Specific
activity (wdmole)
843.5 8640 13000 11510 12090
of RNA
(9020, 7850) (8135, 9145) (14200, 11800) (12025, 10990) (11075, 13110)
a The medium consisted of 0.4 ml of synthetic medium + 0.1 ml of diapause pupal hemolymph plasma. p-Ecdysone dissolved in water was added as indicated. Tissue from pupae in diapause was added and incubated at 25” for 19 hours, then cytidineJH (2.5 &i) was added and incubation was continued for 1 hour. Samples were prepared for counting by procedure B. The means and individual results from two experiments are shown.
Modifications Comparison A B c D
Conk01
- Hemolymph +Ethanol
-
+p-Ecdysone +@-Ecdysone
+Ethanol -
in ethanol in ethanol
+Ethanol
+22-Isoecdysone ethanol
in
Exptl. (E)
E c + SEW
Pb
4985 6025
2280 6063
0.41 0.99
zk 0.01 k 0.14
7 3
4914 5773 ~5172
8231 11503
1.56 1.93
z!z 0.09 * 0.19
9313
1.67
& 0.10
7913 5122 __ 6518
1.41 1.15
+ 0.14 k 0.14
6
5765 -.4359 5062
1.28
f 0.10
<0.05
2
6375
6667
1.05
k 0.05
NS
iii
+a-Ecdysone (combined)
E+F G
+a-Ecdysone +a-Ecdysone
-Control CC)
4 2 in ethanol
+p-Ecdysone (combined)
C+D E F
No. of Expts.
Experimental
+Ethanol
-
to medium
Specific act,ivit? of RKA (cpm/ pmole)
3 3
a The standard medium and conaitions were as described in Table 2. Steroids were used at 5 pg/ml. Ethanol, when used as a solventj or in controls, was 25 ~1 of 10%;. The data from a series of 12 experiments (each comparing four different conditions) are assembled as comparisons of the mean result for each experimental condition with that for the corresponding control. a See footnotes to Table 1.
by procedure A (data not shown) indicated that hemolymph from animals in diapause or development was about equally effective. Its effect was on preservation of celnot on the incorporation lular activity, process, for hemolymph added at the same time as the isotope, after 19 hours incubation of tissue without it, did not stimulate. In all later experiments, hemolymph was included in the medium. Hormonal effect’s were obtained both without added hemolymph (Table 1) and with it (Tables 2 and 3). Since some hemolymph was always carried in the wing lacunae, however, we cannot tell whether the tissue might respond to ecdysone in its complete absence. Comparison B shows that ethanol, in the small amount used as a solvent, had no effect. The stimulatory effect of ,&ecdysone on RNA synthesis, shown in comparisons C and D, is highly significant. In individual experiments, E/C ranged from 1.19 to 2.17. The apparently greater effect of P-ecdysone in ethanol (D) than in water (C) is not
statistically significant (P > 0.1). The results with #cY-ecdysone are shown in comparisons E and F ; in some experiments the ethanol control was omitted since no difference had been found from the water control. The effect of a-ecdysone is less than that of P-ecdysone at the same concentration, and in some experiments no effect was observed (E/C ranged from 0.98 to 1.68). When the 6 experiments with cu-ecdysone are combined (E + F), however, the stimulation is st’atistically significant. Also, comparison of the combined data for a-ecdysone (E + F) with those for /?-ecdysone (C + D) shows that the difference between the effects of the two hormones is significant (2’ < 0.02). 22-Isoecdysone (comparison G) was inactive. DISCUSSION
Since pure ecdysones have become available, cytological and developmental effects of these hormones upon several insect tissues in vitro have been described (review: Wyatt, 1971). Stimulation of RNA syn-
ECDYSOKE
AND
SYNTHESIS
thesis in isolated insect cell nuclei by added ecdysone has also been claimed (Sekeris et al., 1965; Congote et al., 1969). The only biochemical response of an intact insect tissue to ecdysone in vitro yet reported is that of DNA synthesis in imaginal discs of the wax moth (Galleria mellonella), demonstrated by autoradiography (Oberlander, 1969). In the present paper we add quantitative demonstration of stimulation of RNA and protein synthesis by ecdysone added to isolated intact insect tissue. After incubating wing tissue from Cecropia in diapauee for 20 hours with p-ecdysone at 5 pg/ml, we find rates of RNA and protein synthesis elevated, on the average, about 65% above controls, and there was some positive effect in every experiment. With #a-ecdysone, on the other hand, the stimulation was less (average 30% above controls) and variable, so that in some experiments none was seen. The apparent ineffectiveness of a-ecdysone (2 ,pg/ml) in an experiment reported previously (Retldy and Wyatt, 1967) is therefore not inconsistent wit,h t,he present data. The specificity of the effect is further shown by the inactivity of 22-isoecdyeone, an analog which is inert in other ecdysoneresponsive systems (Furlenmeier et al.. 1967; .Judy, 1969; Marks and Leopold, 1970). The greater effect of /3- than cy-ecdysone is interesting in relation to the ouestion of their respective biological roles. Although a-ecdysone was the first to be isolated in pure form from an insect source, recent work indicates that insects generally contain more fi-e?dysone (review: Wyatt, 1971). Also, rapid conversion of cr- to /3ecdysone (20-hydroxylation) in insects has been demonstrated (King and Siddall, 1969; Moriyama et al., 1970). It is thus quite likely that P-ecdysone is the more generally active form of the hormone. On the other hand, DNA synthesis in Gal&a wing discs in vitro is reported to respond to a-ecdysone but not to ,&ecdyeone (Oberlander, 1969). Comparison of dose and time relationships in the action of the two ecdvsones in vitro should clarifv their roles. The increase in hiosynt,hesis after in-
IN
MOTH
WINGS
373
cubation of tissue with p-ecdysone for 20 hours is comparable to that found in tissue from animals in which the hormone had been injected 20 hours earlier (Fig. 1). Both, however, are much smaller than the elevation found by measurement in vivo after a similar time of ecdysone action, when incorporation int’o both RNA and protein may be elevated as much as IO-fold (Wyatt, 1970). The smaller apparent hormonal stimulation in vitro may be due to increased activity in the control tissue as a result of stripping from the pupal cuticle, as has been pointed out previously (Reddy and Wyatt, 1967). The stimulatory effect of ecdysone on cecropia wing tissue encourages us to continue the biochemical study of hormonal action on insect tissues in vitro. ACKNOWLEDGMENTS We are grateful J. B. Siddall for gifts work was supported Foundation and the U. S. Public Health
to
Dr. 9. D. Cross and Dr. of synthetic hormones. This by grants from the Whitehall National Institutes of Health, Service (HD-02176).
REFERENCES L. F., SEKERIS, C. E., .~ND KARLSON, P. (1969). On the mechanism of hormone action. XIII. Stimulating effects of ecdysone, juvenile hormone, and ions on RNA synthesis in fat body cell nuclei from Culliphora erythrocephaln isolated by a filtration technique. Exp. Cell Res. 56, 338-346. FURLENMEIER, A., F~~RsT. A.. 1J~~~~~~4~~. -4., WPILDVOGEL: G., HOCKS. P.. KERB, U.. AND WEICHERT, R. (1967). Zur Synthese des Ecdysons. IX. Mitteilung iiber Insektenhormone. Hefv. Chim. Actn 50, 2387-2396. GORELL, T. A.. AND GILBZRT. I,. I. (1969). Stimulation of protein and R,NA synthesis in the crapfish hepatopancreas by crustecdysone. Gen. Comp. Endocrinol. 13, 308-310. HOWELLS, A. M., AND WYATT, G. R. (1969). The apparent template activity of RNA from developing wings of the cecropia silkmoth. Biochim. Biophys. Acta 174, 86-98. KARLSON. P., AND SEKERIS. C. E. (1966). Ecdysone. an insect steroid hormonr. and its mode of action. Recent Progr. Helm. Res. 22, 473-502. KING, D. S.. AND SIDDALL, J. B. (1969). Conversion of cr-ecdysone to P-ecdysonc by crustaceans and insects, Nntxre (London) 221, 956956. CONGOTE,
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JUUY. K. J. (1969). Cellular resl,onsc: to c~c++ terone itl uitro. Science 165, 137441375. LUCAS. K. U. (1968). DSA-RNA hybridization studies on the developing wings of thcx c,ccropia silkmoth. Ph.D. dirsertation. Yale I’nitersity. New Haven. Connecticut. MARKS, E. P., AND LEOPOLD, R. A. (1970). Cockroach leg regeneration: effects of ec*dyst,erone in vitro. Science 167, 61-62. MORIYAMA, H.. NAKANISHI, K.. KING, D. S.. OKAUCHI, T., SIDDALL, J. B.. AND HAFFERL, W. (1970). On the origin and metabolic fate of a-ecdysone in insects. Gen. Comp. Endocrinol., 15, 80. MUNRO. H. N., AND FLECK, A. (1966). The determination of nucleic acids. Metk. Biochem. Anal. 14, 113-176. NEUFELD, G. J., THOMSON, J. A., AND HORN, D. S. H. (1968). Short-term effects of crustecdysone (20-hydroxyecdysone) on protein and RNA synthesis in third-instar larvae of Calliphora. J. Insect Physiol. 14, 789-802. OBERLANDER, H. (1969). Ecdysone and DN.4 synthesis in cultured wing disks of the was moth, Galleria mellonellx J. Insect Physiol. 15, 18031806. REDDY, S. R. R.. AND WYATT, G. R. (1967). Incorporation of uridine and leucine in vitro by cecropia silkmoth wing epidermis during diapause and development. J. Insect Physiol. 13, 981-994. RIDDIFORD, L. M. (1968). An artificial diet for
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H. I-\.. .\~a RILLIIMS. C. IQ. (1953). The physiology of insf,ct di:cl~xu~i’~. VII. The resl+:itory met.aboli+m of the I.ecropia silkworm during diapauac and develol~ment. Biol. Bull. 105, 320-334. SEKERIS. C. l3., DUKES. P. P., ASD S;(IHMII). W. (1965). Wirkung von Ecdyson auf Flpidcrmiszellkerne von Cnlliphom I,ar\-cn i)t ~~ih. IIoppeSeyler’s Z. Phusiol. Chenz. 341, 152-154. STEVENSON, E.. AND wy.4~~. G. R. (1962). The metabolism of silkmoth tissues. Incorporation of leucine into protein. Arch. Biochenl. Biophys. 99, 65-71. TATA. J. R. (1966). Hormones and the synthesis and utilization of ribonucleic acids. Progr. N~cl. Acid RPS. Mol. Biol. 5, 191-250. WILLIAMS. C. M. (1946). Physiology of insect diapause: the role of the brain in the production and termination of pupal dormanq in the giant silkworm. P. cecropin. Biol. Bull. 90, 234243. WYATT, G. R. (1967). Macromolecular hiosynthesis in insect metamorphosis. Inl. (‘ongr. Biochem., 7th, Tokyo, Abstr. 2. 389. WYATT. G. R. (1970). Nucleic acid and protein synthesis in hormone-induced development in silkmoth pupae. In preparation. WYATT. G. R. (1971). Insect hormones. In “Biochemical Actions of Hormones” (G. Litwack. ed.), Vol. 2. Academic Press, New York. (In press.) SVIISEII~ISHX~S.