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Vitellogenin synthesis by the fat body of the mosquito Aedes aegypti
DEVELOPMENTAL
BIOLOGY
.? t,
Vitellogenin
285-294
(1973)
Synthesis
by the Fat Body of the Mosquito A edes aeg yp ti:
Evidence
for Transcriptional
Control’
H. H. HAGEDORN,~ANN MARIE FALLON,’ AND HANS LAUFER Biological
Sciences Group, University Accepted
of Connecticut,
Storrs, Connecticut
06268
October 6, 197.2
Synthesis of vitellogenin (yolk protein) by the fat body of Aedes aegypti is triggered by the blood meal. Total RNA of the fat body begins to rise 2 hr post blood meal (PBM) and increases 3-fold by 12 hr. Vitellogenin synthesis is detectable 3-4 hr PBM, and reaches a peak by 28 hr PBM. After 28 hr PBM both total RNA and the ability to synthesize vitellogenins fall precipitously. Actinomycin D at 10 pglml inhibits RNA synthesis by about 90%, but does not inhibit in uitro synthesis of tissue proteins. At this concentration of actinomycin, vitellogenin synthesis remains constant in vitro for up to 6 hr, suggesting the presence of a relatively long-lived messenger RNA. When injected into mosquitoes, actinomycin prevents the normal increase in the rate of vitellogenin synthesis but allows synthesis to proceed at the rate occurring at the time of injection. The results suggest that the blood meal triggers the synthesis of both messenger and ribosomal RNA necessary for later vitellogenin synthesis.
genie proteins by the fat body, and (2) the uptake of these proteins by the oocyte. The aim of this investigation is to examine the role of the fat body in egg development. The present paper considers some of the events occurring in the fat body after the blood meal and their response to actinomycin D. Future communications will deal with the role of humoral factors in activating the fat body. It is hoped that this work will contribute toward the understanding of the hormonal control of protein synthesis.
INTRODUCTION
The fat body of insects has been shown to synthesize proteins which are sequestered by the developing oocyte (Pan et al., 1969; Engelmann, 1969; Brookes, 1969; Hagedorn and Judson, 1972). These proteins, which are sex limited and selectively removed from the hemolymph by the oocyte (Telfer, 1954, 1960; Roth and Porter, 1964; Anderson, 1964; Stay, 1965; Bell, 1970; Anderson and Telfer, 1970; Anderson and Spielman, 1971) will be termed the vitellogenic proteins (Pan et al., 1969). In Aedes aegypti the blood meal triggers egg development, and since repeated blood meals can be taken, each usually producing a batch of eggs, one would expect that this process is precisely regulated. There are at least two levels at which egg development can be regulated. These are (1) the synthesis of the vitello-
MATERIALS
285 0 1973 by Academc Press. Inc. of remoduction in anv form reserved.
METHODS
Mosquitoes. Mosquitoes were from a laboratory colony of Aedes aegypti of the Liverpool strain reared by standard techniques (Judson, 1967). Adults were held at 22°C until used. Mosquitoes used in experiments were 5-10 days old and were held at 27°C for the duration of the experiment. Organ culture. The method of fat body organ culture is described by Hagedorn and Judson (1972) and will be summarized
’ Supported in part by grants from the National Science Foundation (Grant no. GB 18606) and the University of Connecticut Research Foundation. ’ Present address: Department of Biology, Yale University, New Haven, Connecticut. Copyright All riehts
AND
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here. The fat body was dissected from the female leaving it attached to the abdominal wall; all other tissues, except the epidermis, were removed. The abdominal wall was then split down one side and floated with the fat body hanging free in the medium. The organ culture medium was developed from the salts and glucose mixture used for Aedes aegypti tissue culture (Varma and Pudney, 1969) and the amino acid mixture used for cecropia fat body organ culture (Reddy and Wyatt, 1967). Fat bodies from 10 females were incubated in 0.1 ml of medium for varying periods in spot plate depression slides on a rotating table. At the end of incubation the medium was withdrawn and the tissues were washed twice with 0.05 ml of medium. The fat body was then removed and frozen. The medium was combined with the washings (total volume = 0.2 ml) and centrifuged at 12,500 g for 10 min. To 0.05-ml aliquots of the supernatant were added 0.01 ml of carrier yolk protein (1 mg/ml) and 0.05 ml of antiserum to purified yolk protein. This amount of antiserum was found by preliminary experiments to precipitate all the yolk protein antigens from the medium. The antiserum was absorbed with an extract of males and unfed females before use. The antibody-antigen precipitate was then collected on 0.45 p Millipore filters (presoaked in buffer containing phenylalanine and bovine serum albumin), washed with 40 ml of phosphate-buffered saline, 2 ml of chloroform : methanol (1: I), dried, and counted in a liquid scintillation counter. The fat bodies were homogenized in 0.2 ml Tris ‘Cl pH 8.5 buffer, centrifuged at 12,500 g for 10 min, and total protein was determined on the supernatant by the method of Lowry et al. (1951). The results are expressed as the amount of antibody precipitable label in the medium per microgram of protein in the fat body tissue. The average amount of protein in the fat body preparations from 10 females was 0.5 mg.
VOLUME 31, 1973
Analytical techniques. For determination of total RNA the tissue was homogenized in 95% ethanol containing 10% potassium acetate. The precipitate was washed twice with ether and 5 times with 0.2 N perchloric acid. The extensive washing with perchloric acid removed substances that interfered with the spectrophotometric determination (Linzen and Wyatt, 1964). Hydrolysis was carried out in 0.4 ml of 0.3 N KOH as described by Munro and Fleck (1966). The extracted RNA had a 260:230 ratio > 2. DNA determinations were made on the pellet remaining after hydrolysis of the RNA using the method of Giles and Meyer (1965). Specific activity of RNA labeled with uridine- 3H was determined by extracting the RNA as described above and taking duplicate aliquots for determination of total RNA at 260 nm and of radioactivity by liquid scintillation counting. The results are expressed as dpm/pg RNA. Incorporation of phenylalanine- 3H into soluble fat body tissue proteins was determined by removing the fat bodies at the designated times and homogenizing them in a 0.05 M Tris ‘Cl buffer, pH 8.5 containing 0.15 M NaCl. The homogenate was centrifuged at 12,500 g for 10 min. Duplicate aliquots of the supernatant were taken and the proteins were precipitated with 10% TCA. The precipitate was heated to 100°C for 15 min, washed with ethanol and ether, dried, and then dissolved in 0.2 N KOH. Samples were then analyzed for protein (Lowry et al., 1951) and radioactivity using liquid scintillation counting and NCS (Nuclear Chicago) to solubilize the sample. The results are expressed as dpmlpg protein. Actinomycin D was obtained from Calbiochem. Experiments using actinomycin were carried out in the dark. L-Phenylalanine-3H, (specific activity 4.5 Cilmmole) and uridine-3H (specific activity 6.5 Ci/ mmole) were obtained from New England Nuclear. The L-phenylalanine-3H (specific activity 3.48 Ci/mmole) for the experiment
HAGEDORN, FALLON, AND LAUFER
shown in Fig. 2 and L-phenylalanine-‘*C (specific activity 0.51 Ci/mmole) for the experiment shown in Table 1 were from Amersham/Searle. RESULTS
Kinetics in Vivo
of
Protein
and RNA Synthesis
Changes in the total RNA of the fat body after the blood meal are shown in Fig. 1. The RNA begins to increase about 2 hr post blood meal (PBM) and levels off between 12 and 28 hr. The DNA content of the fat body did not change during this time (Fig. 1). Thus it appears that the increase in RNA occurs without prior DNA replication. It is also apparent that the decline in total RNA between 28 and 36 hr is due not to cell loss, but rather to destruction of intracellular RNA. The ability of the fat body to synthesize vitellogenin (antibody-precipitable material) shows a similar rise and fall (Fig. 2) which is closely synchronized with the change in total RNA: vitellogenin synthesis begins 3-4 hr PBM, reaches a peak by about 28 hr and falls to low levels by 40 hr. The period of 2-3 hr PBM will be termed the “activation period.”
Vitellogenin
Synthesis
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287
We were interested in the events occurring in the fat body during the activation period. Specifically, we wanted to know whether vitellogenin syr&hesis in the fat body is under transcriptional or translational control. That is, is the vitellogenin synthesized messenger RNA (mRNA) prior to the blood meal, stored, and then translated after the blood meal, or does the blood meal trigger synthesis of the vitellogenin mRNA? Actinomycin D, which inhibits synthesis of both mRNA and ribosomal RNA (Tata, 1966), can be used to gain insight into the regulation of protein synthesis. Its use, however, must be carefully controlled (Gross, 1968). The following experiments were designed to provide the controls necessary for an investigation of the effects of actinomycin D on vitellogenin synthesis. Kinetics of Protein in Vitro
and RNA
Synthesis
The viability of the fat body in culture was determined by investigating the kinetics of protein and RNA synthesis. The secretion of labeled vitellogenin is linear in vitro for up to 6 hr (Hagedorn and Judson, 1972). Incorporation of phenylalanine into
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FIG. 1. Change in total RNA and DNA with time after the blood meal. Each point represents the mean of duplicate samples taken from two replicates of 10 fat bodies each. Inset shows a separate experiment for the early time points. Average variation from the mean for the RNA data = 3.5%; for the DNA data = 6%. 0, RNA; W, DNA.
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tissue proteins is linear for at least 60 min after a short lag (Fig. 3). TCA-soluble counts reached an equilibrium within 10 min. The kinetics of uridine incorporation into RNA are shown in Fig. 4. Incorporation increases for about 120 min and then levels off, perhaps reflecting a balance between synthesis and degradation. The fat bodies used in these experiments were removed at 18 hours PBM at which time no increase in total RNA occurs in uiuo (Fig. 1). The presence or absence of 0.025 mM unlabeled uridine (Wyatt and Wyatt, 1971) in the medium did not change the results; thus the decline in rate of incorporation was probably not due to exhaustion of exogenous uridine. However, we have not looked at the specific activity of intracellular uridine during this time period. Reddy and Wyatt (1967) found a similar curve of in vitro uridine incorporation into cecropia wing epidermis. The time required for synthesis and release of vitellogenin during a pulse of phenylalanine-3H followed by a chase with unlabeled phenylalanine is shown in Fig. 5. Antibody-precipitable label appears
VOLUME 31, 1973
FIG. 3. Kinetics of incorporation of phenylalanine into soluble tissue proteins. The fat body was removed 18 hr after feeding and incubated for the indicated times in 0.1 ml of medium containing 5 &i of phenylalanine-3H. Each point represents the mean of duplicate samples taken from two replicates of 5 fat bodies each. Vertical dashed lines indicate the range.
within 30 min, and a stable rate of incorporation is reached by 60 min; during the chase most of the labeled vitellogenin is secreted within 60 min. There appears to be no storage of the vitellogenin after synthesis. These results support the data of Brookes (1969) showing rapid synthesis
+K2;, ,I:, , ,t 24 Hours
FIG. 2. Ability of the fat body meal. The fat body was dissected 5 &i of phenylalanine-3H. Each 10 fat bodies each. The samples indicate the standard error.
30 36 After
42 Blood
48 54 Meal
60
GG
72
to synthesize and secrete labeled vitellogenin at various at the indicated times and incubated for 3 hr in 0.1 ml point represents the mean of duplicate samples taken taken at 18, 24, 28, and 32 hr were replicated 4 times.
times after the blood of medium containing from two replicates of Vertical dashed lines
HAGEDORN,
FALLON,
Vitellogenin
AND LAUFER
Synthesis
in the Mosquito
289
30.
15 30
GO
150 120 MINUTES
210
240
FIG. 4. Kinetics of incorporation of uridine into RNA by fat body in uitro. The fat body was removed at 18 hr after feeding and incubated in 0.1 ml of media containing 5 &i of uridine-3H and 0.025 M unlabeled uridine. Each noint renresents the mean of dunlicate samples taken from two replicates of 5 fat bodies each. Vertical dashed lines indicate the range.
and release of vitellogenin by Leucophaea fat body in vitro. Thus we can conclude that the fat body is viable in vitro for short-term experiments. Although linear incorporation of amino acids into vitellogenin has been demonstrated for 6 hr (Hagedorn and Judson, 1972), we usually incubated the tissues for 3 hr. Under these conditions, then, it is possible to study the effects of actinomytin D on vitellogenin synthesis. Effect of Actinomycin
D in Vitro
In a preliminary experiment we found that actinomycin enters the cells of the fat body and depresses RNA synthesis within 20 min. The effects of various concentrations of actinomycin on the in vitro incorporation of uridine-3H into RNA, and phenylalanine- 14C into soluble tissue proteins are shown in Table 1. Actinomycin at 10 pg/ml inhibits incorporation of uridine by about 87%, or, in another experiment, by 92%. The residual incorporation unaffected by actinomycin may be due, in part, to end-labeling of the terminal CCA
sequence of transfer RNA (Gross and Cousineau, 1964; Gross et al., 1964). At 10 pg/ml actinomycin has very little effect on incorporation of phenylalanine14C into tissue proteins. At concentrations above 40 pg/ml, actinomycin inhibits incorporation into tissue proteins. High concentrations of actinomycin have been shown to interfere with cellular functions in some way unrelated to inhibition of RNA synthesis. For example, actinomycin has been found to affect glycolysis (Honig and Rabinovitz, 1966), concentrations of ATP (Laszlo et al., 1966), phospholipid synthesis (Pastan and Friedman, 1968), and the specific activity of intracellular pools (Wilson and Hoagland, 1967; Regier and Kafatos, 197 1). From these experiments it is evident that actinomycin, at doses which inhibit RNA synthesis by about 90%, has little effect on incorporation of phenylalanine into fat body proteins. We next investigated the kinetics of the effect of actinomycin on vitellogenin synthesis in uitro. Table 2 shows that the rate of incorporation into vitellogenin is essen-
DEVELOPMENTALBIOLOGY
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VOLUME 31. 1973
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FIG. 5. Kinetics of vitellogenin synthesis by the fat body in vitro. The fat body ing and the rate (cpm/lO min) of appearance of antibody-precipitable label in the a pulse of phenylalanine-3H (10 pCilO.1 ml) followed by a chase with unlabeled was replaced every 10 min. Each point represents the mean of samples taken bodies each.
tially constant for 6 hr in the presence of 10 pg/ml of actinomycin. These data suggest that there is no detectable rate of breakdown of vitellogenin mRNA during the 6-hr incubation period. Effects of Actinomycin
in Viuo
Having found that actinomycin has little effect on vitellogenin synthesis in uitro, we injected various concentrations of the drug into mosquitoes to see what effects it has on RNA synthesis in vivo. Table 3 shows that injecting 0.1 /lg per mosquito inhibits 87% of the incorporation of uridine into
was removed 18 hr after feedmedium was measured during phenylalanine. The medium from two replicates of 10 fat
RNA. This concentration was used in the following experiments. Actinomycin was injected at various times after the blood meal and the ability of the fat body to synthesize vitellogenin was assayed in vitro at 18 hr PBM (Table 4). If actinomycin is injected immediately after the activation period (5 hr PBM) the synthesis of vitellogenin is inhibited by over 90%. If actinomycin is injected 12 hr PBM and the synthesis of vitellogenin is assayed 6 hr later, the level of vitellogenin synthesis is that expected for 1%hr rather than for 18 hr (Table 4). The same type of
response is obtained when the mosquitoes are injected at 10 hr PBM. It appears, therefore, that inhibition of RNA synthesis at any time after the activation period
Control 1 10 20 40 60 100
H3 RNA (dpmi~g) 2226 589 300 277 224 165 208
“C Tissue protein-TCA ppt (dpm/d 45.5 46.1 32.1 40.7 38.8 21.2 8.4
“The fat body was removed at 18 hr after feeding and preincubated for 30 min in 0.1 ml of medium containing various concentrations of actinomycin. Uridine-3H (10 &i) and phenylalanine-“C (5 &i) were then added and the incubation was continued for 4 hr. The specific activity of the RNA was determined as described in the text. The specific activity of the tissue protein was determined using the pellet remaining after RNA extraction. Each number represents the mean of duplicate samples taken from 2 or 3 replicates of 5 fat bodies each.
TABLE 2 RATE OF in Vitro VITELLOCENIN SYNTHESIS IN THE PRESENCE AND ABSENCE OF ACTINOMYCIN D” Hour
Cpm antibody ppt/pg fat body protein/hour Control
1 2 3 4 5 6
39.4 41.7 58.1 56.6 55.8 36.9
Actinomycin (10 &nl)
TABLE 3 EFFECT OF INJECTED ACTINOMYCIN D ON URIDINE INCORPORATIONBY WHOLE MOSQUITOES in uivo” Actinomycin injected/n (ILId Saline control 0.001 0.01 0.1”
TABLE 1 EFFECT OF ACTINOMYCIN D ON in Vitro INCORPORATIONOF PHENYLALANINE-“C INTO PROTEIN AND URIDINE-3H INTO RNA” Actinomycin D concentration Wml)
291
Vitellogenin Synthesis in the Mosquito
HAGEDORN, FALLON, AND LAUFER
D
50.0 73.0 65.2 12.3 64.4 76.0
“The fat body was removed at 18 hr after feeding and incubated with phenylaIanine-aH (50 j&i/ ml) and actinomycin at 10 @g/ml. The medium was replaced every hour and the presence of vitellogenin was assayed as described in the text. Each number represents the mean of samples taken from two replicates of 10 fat bodies each.
Incorporation/n (cpm) 2565 3750 3622 429
“Unfed mosquitoes were injected with 1 ~1 of buffer containing various concentrations of actinomycin D and 0.25 &i of uridine-3H. Two hours later they were homogenized and centrifuged. RNA was precipitated from the supernatant with cold 10% TCA. Each number represents the mean of duplicate samples taken from two replicates of 5 females each. bThe approximate volume of a mosquito is 0.01 ml; therefore the final concentration of actinomycin was about 10 rg/ml.
stops vitellogenin synthesis at the level expected if the fat body had been assayed at the time of injection. Thus the bursts of RNA and vitellogenin synthesis which occur after the activation period are causally related. DISCUSSION
The purpose of this paper is to further investigate the nature of the events occurring in the fat body of the mosquito Aedes aegypti after a blood meal. It has previously been shown that the fat body synthesizes the vitellogenic proteins which are sequestered by the developing oocyte and that the fat body of an unfed female is unable to synthesize vitellogenin (Hagedorn and Judson, 1972). There is also a short time after the blood meal during which the fat body of the fed female does not synthesize vitellogenins (Fig. 2). This time has been designated the “activation period.” The first event we have seen in the fat body is an increase in total RNA, beginning about 2 hr after the blood meal and culminating 12 hr later in a tripling of the fat body RNA. Three hours after the blood meal the fat body begins to secrete vitellogenin. By 28 hr after the blood meal both
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EFFECT OF ACTINOMYCIN D ON SYNTHESIS OF VITELLOGENIN WHEN INJECTED in Viuo”
”
Saline injected 5 hr PBMb assayed 18 hr PBM Saline injected unfed assayed 15 hr post injection Actinomycin injected 5 hr PBM assayed 18 hr PBM Uninjected assayed 5 hr PBM Actinomycin injected 12 hr assayed 18 hr PBM Uninjected assayed 12 hr PBM
Cpm antibody pptlpg fat body protein/hour
2
92.4
2
8.6
4
11.2 + 1.6
2
15.8
5
58.8 * 7.4
4
69.6 i 9.6
” Actinomycin (0.1 wg/ml) was injected at various times after the blood meal. The fat body was removed 18 hr PBM and incubated in medium containing actinomycin D (10 @g/ml) and phenylalanine-3H (50 pCi/ml) for 4 hr. The control tissues were incubated in medium without actinomycin. The data are from three independent experiments. Each number represents the mean of duplicate samples taken from replicates of 10 fat bodies each + the standard error when n > 2. b Post blood meal.
the total RNA of the fat body and its ability to synthesize vitellogenin begin to fall precipitously. We have also found that injection of actinomycin in uiuo at various times after the blood meal prevents the normal increase in vitellogenin synthesis, but allows synthesis to proceed at the rate expected for tissues assayed at the time of injection. This concept is presented diagrammatically in Fig. 6. When the kinetics of vitellogenin synthesis in the presence of actinomycin were examined, the rate of synthesis was constant for 6 hr. This implies that, once synthesized, the vitellogenin mRNA is relatively stable. With the aid of the above data the probable events occurring in the fat body after the blood meal can be reconstructed as follows: the blood meal triggers some un-
VOLUME
31, 1973
known mechanism which results, 2 hr later, in the activation of RNA synthesis. Both ribosomal RNA and vitellogenin mRNA are synthesized, resulting in a tripling of the total RNA of the cell and rapid elaboration of protein synthetic machinery. The vitellogenin mRNA is stable, hence it accumulates, and beginning 3-4 hr after the blood meal the fat body begins to synthesize vitellogenins. The rate of vitellogenin synthesis rises until at 28 hr after the blood meal the RNA of the fat body is broken down and synthesis of vitellogenin declines. This reconstruction is speculative and is presented as a working hypothesis postulating transcriptional control of vitellogenin synthesis. How well do the present data agree with the hypothesis? A critical question relates to the kinds of RNA made during the increase in total RNA. We do not have direct evidence on this point but the results do suggest an answer.
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FIG. 6. The effect of in uiuo injection of actinomytin D on vitellogenin synthesis as measured in vitro. A diagrammatic representation of Fig. 2, redrawn to emphasize the early time points, and the data from Table 4. Arrows indicate time of actinomycin injection, -*time of assay, - - -, level of synthesis at 5 hours, level of synthesis at 12 hr.
HAGEDORN, FALLON, AND LAUFER
If we look first at Fig. 1 it is apparent that the total RNA of the fat body triples from 2 to 12 hr after feeding: this is almost certainly due primarily to accumulation of ribosomal RNA. If the ability to synthesize vitellogenin were limited by the availability of ribosomes, vitellogenin synthesis should also plateau at 12 hr. Figure 2, however, shows that vitellogenin synthesis continues to rise for another 16 hr. Thus it appears that during the first 12 hr both mRNA and the protein synthetic machinery are developed simultaneously. It is likely that after this period the increased rate of synthesis is due solely to continued synthesis and accumulation of vitellogenin mRNA. Several questions can be posed using the above model. One of these involves the nature of the “unknown mechanism” which triggers RNA synthesis. Hormones are certainly involved as the work of Clements (1956), Gillett (1956, 1957), Larsen (1958), Larsen and Bodenstein (1959), and Lea (1967) indicates. Recent work in the field of hormonal control of protein synthesis in other systems indicates that protein synthesis can be stimulated either without prior RNA synthesis or with an obligatory synthesis of RNA before protein synthesis begins (Schmike and Doyle, 1970; Lee et al., 1970). The hormonal control of vitellogenin synthesis in the mosquito is apparently of the latter type. Recent work in this laboratory has demonstrated that the ovary is the source of a humoral factor involved in activating the fat body (Hagedorn and Fallon, in preparation). Other questions one might pose to test the hypothesis of transcriptional control relate to identifying the kinds of RNA being made after the activation period and the nature of the mechanisms involved in the decline of vitellogenin synthesis after 28 hr. The unique aspects of this system which have been outlined here provide an unusual opportunity to study the hormonal
Vitellogenin
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Thanks are due to James P. Calvet and Lee Weber for stimulating discussions during the course of this work. REFERENCES ANDERSON, E. (1964). Oocyte differentiation and vitellogenesis in the roach Periplaneta americana. J. Cell Biol. 20, 131-155. ANDERSON, L. M., and TELFER, W. H. (1970). Trypan blue inhibition of yolk deposition-a clue to follicle cell function in the cecropia moth. J. Embryol. Exp. Morphol. 23, 35-52. ANDERSON, W. A. and SPIELMAN, A. (1971). Permeability of the ovarian follicle of Aedes aegypti mosquitoes. J. Cell Biol. 50, 201-221. BELL, W. J. (1970). Demonstration and characterization of two vitellogenic blood proteins in Periplaneta americana: an immunochemical analysis. J. Insect Physiol. 16, 291-299. BROOKES, V. J. (1969). The induction of yolk protein synthesis in the fat body of an insect, Leucophaea maderae, by an analog of the juvenile hormone. Develop. Biol. 20, 459-471. CLEMENTS, A. N. (1956). Hormonal control of ovary development in mosquitoes. J. Exp. Biol. 33, 211223. ENGELMANN, F. (1969). Female specific protein: Biosynthesis controlled by corpus allatum in Leucophaea maderae. Science 165, 407-409. GILES, K. W. and MEYERS, A. (1965). An improved diphenylamine method for the estimation of deoxyribonucleic acid. Nature (London) 206, 93. GILLETT, J. D. (1956). Initiation and promotion of ovarian development in the mosquito Aedes aegypti. Ann. 7’rop. Med. Parasitol. 50, 375-380. GILI~ETT, J. D. (1957). Variation in the time of release of the ovarian development hormone in Aedes aegypti. Nature (London) 180, 656. GROSS, P. R. (1968). Actinomycin in Developmental Biology. In “Actinomycin, Nature, Formation and Activities” (S. A. Waksman, ed.), pp. 87-100. Wiley, New York. GROSS, P. R., and COLJSINEAU,G. H. (1964). Macromolecule synthesis and the influence of actinomytin on early development. Exp. Cell. Res. 33, 368395. GROSS, P. R., MALKIN, L. I., and MOYER, W. A. (1964). Templates for the first proteins of embryonic development. Proc. Nat. Acad. Sci. U.S. 51, 407-141. HAGEDORN, H. H., and JUDSON, C. L. (1972). Purification and site of synthesis of Aedes aegypti yolk proteins. J. Exp. 2001. 182, 367. HONIG, G. R., and RABINOVITZ, M. (1966). Inhibition by actinomycin D of oxidation dependent biosyn-
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thesis in sarcoma 37 ascites cells. J. Biol. Chem. 241, 1681-1687. JUDSON, C. L. (1967). Feeding and oviposition behavior in the mosquito Aedes aegypti. I. Preliminary studies of physiological control mechanisms. Biol. Bull. 133, 369-377. LARSEN, J. R. (1968). Hormone-induced ovarian development in mosquitoes. Science 127, 587-588. LARSEN, J. R., and BODENSTEIN, D. (1959). The humoral control of egg maturation in the mosquito. J. Exp. 2001. 140, 343-382. LASZLO, J., MILLER, D. S., MCCARTY, K. S., and HOCHSTEIN, P. (1966). Actinomycin D: Inhibition of respiration and glycolysis. Science 151, 10071009. LEA, A. 0. (1967). The medial neurosecretory cells and egg maturation in mosquitoes. J. Insect. Physiol. 13, 419-429. LEE, K., REEL, J. R., and KENNEY, F. T. (1970). Regulation of tyrosine LYketoglutarate transaminase in rat liver. IX. Studies of the mechanisms of hormonal inductions in cultured hepatoma cells. J. Viol. Chem. 245, 5806-5812. LINZEN, B., and WYATT, G. R. (1964). The nucleic acid content of tissues of cecropia silkmoth pupae: Relations to body size and development. Rio&em. Biophys. Acta 87, 188-198. LOWRY, 0. H., ROSEBROUGH,N. J., FARR, A. L., and RANDALL, R. J. (1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265275. MUNRO, H. N. and FLECK, A. (1966). The determination of nucleic acids. In “Methods in Biochemical Analysis” (D. Glick, ed.) Vol. 14, pp. 113-176. Wiley, New York. PAN, M. L., BELL, W. J., and TELFER, W. H. (1969). Vitellogenic blood protein synthesis by insect fat body. Science 165, 393-394. PASTAN, I., and FRIEDMAN, R. M. (1968). Actinomycin D: inhibition of phospholipid synthesis in chick
VOLUME 31, 1973 embryo cells. Science 160, 316-317. REDDY, S. R. R., and WYATT, G. R. (1967). Incorporation of uridine and leucine in uitro by cecropia silkmoth wing epidermis during diapause and development. J. Insect Physiol. 13, 981-994. REIGER, J. C., and KAFATOS, F. C. (1971). Microtechnique for determining the specific activity or radioactive intracellular leucine and application to in vitro studies of protein synthesis. J. Biol. Chem. 246, 6480-6488. ROTH, T. F., and PORTER, K. R. (1964). Yolk protein uptake in the oocyte of the mosquito Aedes aegypti. J. Cell Biol. 20, 303-332. SCHMIKE, R. ‘I’., and DOYLE, D. (1970). Control of enzyme levels of animal tissues. Annu. Rev. Eiohem. 39, 929-976. STAY, B. (1965). Protein uptake in the oocytes of the cecropiu moth. J. Cell Biol. 26, 49-62. TATA, J. R. (1966). Hormones and the synthesis and utilization of ribonucleic acids. Progr. Nucl. Acid Mol. Biol. 5, 191-250. TELFER, W. H. (1954). Immunological studies of insect metamorphosis. II. The role of a sex-limited blood protein in egg formation by the cecropia silkworm. J. Gen. Physiol. 37, 539-558. TELFER, W. H. (1960). The selective accumulation of blood proteins by the oocytes of saturniid moths. Biol. Bull. 118, 338-351. VARMA, M. G. R., and PUDNEY, M. (1969). The growth and serial passage of cell lines from Aedes aegypti larvae in different media. J. Med. Entomol. 6, 432. WILSON, S. A., and HOAGLAND, M. B. (1967). Physiology of rat-liver polysomes: The stability of messenger ribonucleic acid and ribosomes. Biochem. J. 103, 556-566. WYATT, S. S., and WYATT, G. R. (1971). Stimulation of RNA and protein synthesis in silkmoth pupal wing tissue by ecdysone in uitro. Germ. Camp. Endocrinol. 16, 369-374.
BIOLOGY
.? t,
Vitellogenin
285-294
(1973)
Synthesis
by the Fat Body of the Mosquito A edes aeg yp ti:
Evidence
for Transcriptional
Control’
H. H. HAGEDORN,~ANN MARIE FALLON,’ AND HANS LAUFER Biological
Sciences Group, University Accepted
of Connecticut,
Storrs, Connecticut
06268
October 6, 197.2
Synthesis of vitellogenin (yolk protein) by the fat body of Aedes aegypti is triggered by the blood meal. Total RNA of the fat body begins to rise 2 hr post blood meal (PBM) and increases 3-fold by 12 hr. Vitellogenin synthesis is detectable 3-4 hr PBM, and reaches a peak by 28 hr PBM. After 28 hr PBM both total RNA and the ability to synthesize vitellogenins fall precipitously. Actinomycin D at 10 pglml inhibits RNA synthesis by about 90%, but does not inhibit in uitro synthesis of tissue proteins. At this concentration of actinomycin, vitellogenin synthesis remains constant in vitro for up to 6 hr, suggesting the presence of a relatively long-lived messenger RNA. When injected into mosquitoes, actinomycin prevents the normal increase in the rate of vitellogenin synthesis but allows synthesis to proceed at the rate occurring at the time of injection. The results suggest that the blood meal triggers the synthesis of both messenger and ribosomal RNA necessary for later vitellogenin synthesis.
genie proteins by the fat body, and (2) the uptake of these proteins by the oocyte. The aim of this investigation is to examine the role of the fat body in egg development. The present paper considers some of the events occurring in the fat body after the blood meal and their response to actinomycin D. Future communications will deal with the role of humoral factors in activating the fat body. It is hoped that this work will contribute toward the understanding of the hormonal control of protein synthesis.
INTRODUCTION
The fat body of insects has been shown to synthesize proteins which are sequestered by the developing oocyte (Pan et al., 1969; Engelmann, 1969; Brookes, 1969; Hagedorn and Judson, 1972). These proteins, which are sex limited and selectively removed from the hemolymph by the oocyte (Telfer, 1954, 1960; Roth and Porter, 1964; Anderson, 1964; Stay, 1965; Bell, 1970; Anderson and Telfer, 1970; Anderson and Spielman, 1971) will be termed the vitellogenic proteins (Pan et al., 1969). In Aedes aegypti the blood meal triggers egg development, and since repeated blood meals can be taken, each usually producing a batch of eggs, one would expect that this process is precisely regulated. There are at least two levels at which egg development can be regulated. These are (1) the synthesis of the vitello-
MATERIALS
285 0 1973 by Academc Press. Inc. of remoduction in anv form reserved.
METHODS
Mosquitoes. Mosquitoes were from a laboratory colony of Aedes aegypti of the Liverpool strain reared by standard techniques (Judson, 1967). Adults were held at 22°C until used. Mosquitoes used in experiments were 5-10 days old and were held at 27°C for the duration of the experiment. Organ culture. The method of fat body organ culture is described by Hagedorn and Judson (1972) and will be summarized
’ Supported in part by grants from the National Science Foundation (Grant no. GB 18606) and the University of Connecticut Research Foundation. ’ Present address: Department of Biology, Yale University, New Haven, Connecticut. Copyright All riehts
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here. The fat body was dissected from the female leaving it attached to the abdominal wall; all other tissues, except the epidermis, were removed. The abdominal wall was then split down one side and floated with the fat body hanging free in the medium. The organ culture medium was developed from the salts and glucose mixture used for Aedes aegypti tissue culture (Varma and Pudney, 1969) and the amino acid mixture used for cecropia fat body organ culture (Reddy and Wyatt, 1967). Fat bodies from 10 females were incubated in 0.1 ml of medium for varying periods in spot plate depression slides on a rotating table. At the end of incubation the medium was withdrawn and the tissues were washed twice with 0.05 ml of medium. The fat body was then removed and frozen. The medium was combined with the washings (total volume = 0.2 ml) and centrifuged at 12,500 g for 10 min. To 0.05-ml aliquots of the supernatant were added 0.01 ml of carrier yolk protein (1 mg/ml) and 0.05 ml of antiserum to purified yolk protein. This amount of antiserum was found by preliminary experiments to precipitate all the yolk protein antigens from the medium. The antiserum was absorbed with an extract of males and unfed females before use. The antibody-antigen precipitate was then collected on 0.45 p Millipore filters (presoaked in buffer containing phenylalanine and bovine serum albumin), washed with 40 ml of phosphate-buffered saline, 2 ml of chloroform : methanol (1: I), dried, and counted in a liquid scintillation counter. The fat bodies were homogenized in 0.2 ml Tris ‘Cl pH 8.5 buffer, centrifuged at 12,500 g for 10 min, and total protein was determined on the supernatant by the method of Lowry et al. (1951). The results are expressed as the amount of antibody precipitable label in the medium per microgram of protein in the fat body tissue. The average amount of protein in the fat body preparations from 10 females was 0.5 mg.
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Analytical techniques. For determination of total RNA the tissue was homogenized in 95% ethanol containing 10% potassium acetate. The precipitate was washed twice with ether and 5 times with 0.2 N perchloric acid. The extensive washing with perchloric acid removed substances that interfered with the spectrophotometric determination (Linzen and Wyatt, 1964). Hydrolysis was carried out in 0.4 ml of 0.3 N KOH as described by Munro and Fleck (1966). The extracted RNA had a 260:230 ratio > 2. DNA determinations were made on the pellet remaining after hydrolysis of the RNA using the method of Giles and Meyer (1965). Specific activity of RNA labeled with uridine- 3H was determined by extracting the RNA as described above and taking duplicate aliquots for determination of total RNA at 260 nm and of radioactivity by liquid scintillation counting. The results are expressed as dpm/pg RNA. Incorporation of phenylalanine- 3H into soluble fat body tissue proteins was determined by removing the fat bodies at the designated times and homogenizing them in a 0.05 M Tris ‘Cl buffer, pH 8.5 containing 0.15 M NaCl. The homogenate was centrifuged at 12,500 g for 10 min. Duplicate aliquots of the supernatant were taken and the proteins were precipitated with 10% TCA. The precipitate was heated to 100°C for 15 min, washed with ethanol and ether, dried, and then dissolved in 0.2 N KOH. Samples were then analyzed for protein (Lowry et al., 1951) and radioactivity using liquid scintillation counting and NCS (Nuclear Chicago) to solubilize the sample. The results are expressed as dpmlpg protein. Actinomycin D was obtained from Calbiochem. Experiments using actinomycin were carried out in the dark. L-Phenylalanine-3H, (specific activity 4.5 Cilmmole) and uridine-3H (specific activity 6.5 Ci/ mmole) were obtained from New England Nuclear. The L-phenylalanine-3H (specific activity 3.48 Ci/mmole) for the experiment
HAGEDORN, FALLON, AND LAUFER
shown in Fig. 2 and L-phenylalanine-‘*C (specific activity 0.51 Ci/mmole) for the experiment shown in Table 1 were from Amersham/Searle. RESULTS
Kinetics in Vivo
of
Protein
and RNA Synthesis
Changes in the total RNA of the fat body after the blood meal are shown in Fig. 1. The RNA begins to increase about 2 hr post blood meal (PBM) and levels off between 12 and 28 hr. The DNA content of the fat body did not change during this time (Fig. 1). Thus it appears that the increase in RNA occurs without prior DNA replication. It is also apparent that the decline in total RNA between 28 and 36 hr is due not to cell loss, but rather to destruction of intracellular RNA. The ability of the fat body to synthesize vitellogenin (antibody-precipitable material) shows a similar rise and fall (Fig. 2) which is closely synchronized with the change in total RNA: vitellogenin synthesis begins 3-4 hr PBM, reaches a peak by about 28 hr and falls to low levels by 40 hr. The period of 2-3 hr PBM will be termed the “activation period.”
Vitellogenin
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We were interested in the events occurring in the fat body during the activation period. Specifically, we wanted to know whether vitellogenin syr&hesis in the fat body is under transcriptional or translational control. That is, is the vitellogenin synthesized messenger RNA (mRNA) prior to the blood meal, stored, and then translated after the blood meal, or does the blood meal trigger synthesis of the vitellogenin mRNA? Actinomycin D, which inhibits synthesis of both mRNA and ribosomal RNA (Tata, 1966), can be used to gain insight into the regulation of protein synthesis. Its use, however, must be carefully controlled (Gross, 1968). The following experiments were designed to provide the controls necessary for an investigation of the effects of actinomycin D on vitellogenin synthesis. Kinetics of Protein in Vitro
and RNA
Synthesis
The viability of the fat body in culture was determined by investigating the kinetics of protein and RNA synthesis. The secretion of labeled vitellogenin is linear in vitro for up to 6 hr (Hagedorn and Judson, 1972). Incorporation of phenylalanine into
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FIG. 1. Change in total RNA and DNA with time after the blood meal. Each point represents the mean of duplicate samples taken from two replicates of 10 fat bodies each. Inset shows a separate experiment for the early time points. Average variation from the mean for the RNA data = 3.5%; for the DNA data = 6%. 0, RNA; W, DNA.
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tissue proteins is linear for at least 60 min after a short lag (Fig. 3). TCA-soluble counts reached an equilibrium within 10 min. The kinetics of uridine incorporation into RNA are shown in Fig. 4. Incorporation increases for about 120 min and then levels off, perhaps reflecting a balance between synthesis and degradation. The fat bodies used in these experiments were removed at 18 hours PBM at which time no increase in total RNA occurs in uiuo (Fig. 1). The presence or absence of 0.025 mM unlabeled uridine (Wyatt and Wyatt, 1971) in the medium did not change the results; thus the decline in rate of incorporation was probably not due to exhaustion of exogenous uridine. However, we have not looked at the specific activity of intracellular uridine during this time period. Reddy and Wyatt (1967) found a similar curve of in vitro uridine incorporation into cecropia wing epidermis. The time required for synthesis and release of vitellogenin during a pulse of phenylalanine-3H followed by a chase with unlabeled phenylalanine is shown in Fig. 5. Antibody-precipitable label appears
VOLUME 31, 1973
FIG. 3. Kinetics of incorporation of phenylalanine into soluble tissue proteins. The fat body was removed 18 hr after feeding and incubated for the indicated times in 0.1 ml of medium containing 5 &i of phenylalanine-3H. Each point represents the mean of duplicate samples taken from two replicates of 5 fat bodies each. Vertical dashed lines indicate the range.
within 30 min, and a stable rate of incorporation is reached by 60 min; during the chase most of the labeled vitellogenin is secreted within 60 min. There appears to be no storage of the vitellogenin after synthesis. These results support the data of Brookes (1969) showing rapid synthesis
+K2;, ,I:, , ,t 24 Hours
FIG. 2. Ability of the fat body meal. The fat body was dissected 5 &i of phenylalanine-3H. Each 10 fat bodies each. The samples indicate the standard error.
30 36 After
42 Blood
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GG
72
to synthesize and secrete labeled vitellogenin at various at the indicated times and incubated for 3 hr in 0.1 ml point represents the mean of duplicate samples taken taken at 18, 24, 28, and 32 hr were replicated 4 times.
times after the blood of medium containing from two replicates of Vertical dashed lines
HAGEDORN,
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Vitellogenin
AND LAUFER
Synthesis
in the Mosquito
289
30.
15 30
GO
150 120 MINUTES
210
240
FIG. 4. Kinetics of incorporation of uridine into RNA by fat body in uitro. The fat body was removed at 18 hr after feeding and incubated in 0.1 ml of media containing 5 &i of uridine-3H and 0.025 M unlabeled uridine. Each noint renresents the mean of dunlicate samples taken from two replicates of 5 fat bodies each. Vertical dashed lines indicate the range.
and release of vitellogenin by Leucophaea fat body in vitro. Thus we can conclude that the fat body is viable in vitro for short-term experiments. Although linear incorporation of amino acids into vitellogenin has been demonstrated for 6 hr (Hagedorn and Judson, 1972), we usually incubated the tissues for 3 hr. Under these conditions, then, it is possible to study the effects of actinomytin D on vitellogenin synthesis. Effect of Actinomycin
D in Vitro
In a preliminary experiment we found that actinomycin enters the cells of the fat body and depresses RNA synthesis within 20 min. The effects of various concentrations of actinomycin on the in vitro incorporation of uridine-3H into RNA, and phenylalanine- 14C into soluble tissue proteins are shown in Table 1. Actinomycin at 10 pg/ml inhibits incorporation of uridine by about 87%, or, in another experiment, by 92%. The residual incorporation unaffected by actinomycin may be due, in part, to end-labeling of the terminal CCA
sequence of transfer RNA (Gross and Cousineau, 1964; Gross et al., 1964). At 10 pg/ml actinomycin has very little effect on incorporation of phenylalanine14C into tissue proteins. At concentrations above 40 pg/ml, actinomycin inhibits incorporation into tissue proteins. High concentrations of actinomycin have been shown to interfere with cellular functions in some way unrelated to inhibition of RNA synthesis. For example, actinomycin has been found to affect glycolysis (Honig and Rabinovitz, 1966), concentrations of ATP (Laszlo et al., 1966), phospholipid synthesis (Pastan and Friedman, 1968), and the specific activity of intracellular pools (Wilson and Hoagland, 1967; Regier and Kafatos, 197 1). From these experiments it is evident that actinomycin, at doses which inhibit RNA synthesis by about 90%, has little effect on incorporation of phenylalanine into fat body proteins. We next investigated the kinetics of the effect of actinomycin on vitellogenin synthesis in uitro. Table 2 shows that the rate of incorporation into vitellogenin is essen-
DEVELOPMENTALBIOLOGY
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FIG. 5. Kinetics of vitellogenin synthesis by the fat body in vitro. The fat body ing and the rate (cpm/lO min) of appearance of antibody-precipitable label in the a pulse of phenylalanine-3H (10 pCilO.1 ml) followed by a chase with unlabeled was replaced every 10 min. Each point represents the mean of samples taken bodies each.
tially constant for 6 hr in the presence of 10 pg/ml of actinomycin. These data suggest that there is no detectable rate of breakdown of vitellogenin mRNA during the 6-hr incubation period. Effects of Actinomycin
in Viuo
Having found that actinomycin has little effect on vitellogenin synthesis in uitro, we injected various concentrations of the drug into mosquitoes to see what effects it has on RNA synthesis in vivo. Table 3 shows that injecting 0.1 /lg per mosquito inhibits 87% of the incorporation of uridine into
was removed 18 hr after feedmedium was measured during phenylalanine. The medium from two replicates of 10 fat
RNA. This concentration was used in the following experiments. Actinomycin was injected at various times after the blood meal and the ability of the fat body to synthesize vitellogenin was assayed in vitro at 18 hr PBM (Table 4). If actinomycin is injected immediately after the activation period (5 hr PBM) the synthesis of vitellogenin is inhibited by over 90%. If actinomycin is injected 12 hr PBM and the synthesis of vitellogenin is assayed 6 hr later, the level of vitellogenin synthesis is that expected for 1%hr rather than for 18 hr (Table 4). The same type of
response is obtained when the mosquitoes are injected at 10 hr PBM. It appears, therefore, that inhibition of RNA synthesis at any time after the activation period
Control 1 10 20 40 60 100
H3 RNA (dpmi~g) 2226 589 300 277 224 165 208
“C Tissue protein-TCA ppt (dpm/d 45.5 46.1 32.1 40.7 38.8 21.2 8.4
“The fat body was removed at 18 hr after feeding and preincubated for 30 min in 0.1 ml of medium containing various concentrations of actinomycin. Uridine-3H (10 &i) and phenylalanine-“C (5 &i) were then added and the incubation was continued for 4 hr. The specific activity of the RNA was determined as described in the text. The specific activity of the tissue protein was determined using the pellet remaining after RNA extraction. Each number represents the mean of duplicate samples taken from 2 or 3 replicates of 5 fat bodies each.
TABLE 2 RATE OF in Vitro VITELLOCENIN SYNTHESIS IN THE PRESENCE AND ABSENCE OF ACTINOMYCIN D” Hour
Cpm antibody ppt/pg fat body protein/hour Control
1 2 3 4 5 6
39.4 41.7 58.1 56.6 55.8 36.9
Actinomycin (10 &nl)
TABLE 3 EFFECT OF INJECTED ACTINOMYCIN D ON URIDINE INCORPORATIONBY WHOLE MOSQUITOES in uivo” Actinomycin injected/n (ILId Saline control 0.001 0.01 0.1”
TABLE 1 EFFECT OF ACTINOMYCIN D ON in Vitro INCORPORATIONOF PHENYLALANINE-“C INTO PROTEIN AND URIDINE-3H INTO RNA” Actinomycin D concentration Wml)
291
Vitellogenin Synthesis in the Mosquito
HAGEDORN, FALLON, AND LAUFER
D
50.0 73.0 65.2 12.3 64.4 76.0
“The fat body was removed at 18 hr after feeding and incubated with phenylaIanine-aH (50 j&i/ ml) and actinomycin at 10 @g/ml. The medium was replaced every hour and the presence of vitellogenin was assayed as described in the text. Each number represents the mean of samples taken from two replicates of 10 fat bodies each.
Incorporation/n (cpm) 2565 3750 3622 429
“Unfed mosquitoes were injected with 1 ~1 of buffer containing various concentrations of actinomycin D and 0.25 &i of uridine-3H. Two hours later they were homogenized and centrifuged. RNA was precipitated from the supernatant with cold 10% TCA. Each number represents the mean of duplicate samples taken from two replicates of 5 females each. bThe approximate volume of a mosquito is 0.01 ml; therefore the final concentration of actinomycin was about 10 rg/ml.
stops vitellogenin synthesis at the level expected if the fat body had been assayed at the time of injection. Thus the bursts of RNA and vitellogenin synthesis which occur after the activation period are causally related. DISCUSSION
The purpose of this paper is to further investigate the nature of the events occurring in the fat body of the mosquito Aedes aegypti after a blood meal. It has previously been shown that the fat body synthesizes the vitellogenic proteins which are sequestered by the developing oocyte and that the fat body of an unfed female is unable to synthesize vitellogenin (Hagedorn and Judson, 1972). There is also a short time after the blood meal during which the fat body of the fed female does not synthesize vitellogenins (Fig. 2). This time has been designated the “activation period.” The first event we have seen in the fat body is an increase in total RNA, beginning about 2 hr after the blood meal and culminating 12 hr later in a tripling of the fat body RNA. Three hours after the blood meal the fat body begins to secrete vitellogenin. By 28 hr after the blood meal both
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4
EFFECT OF ACTINOMYCIN D ON SYNTHESIS OF VITELLOGENIN WHEN INJECTED in Viuo”
”
Saline injected 5 hr PBMb assayed 18 hr PBM Saline injected unfed assayed 15 hr post injection Actinomycin injected 5 hr PBM assayed 18 hr PBM Uninjected assayed 5 hr PBM Actinomycin injected 12 hr assayed 18 hr PBM Uninjected assayed 12 hr PBM
Cpm antibody pptlpg fat body protein/hour
2
92.4
2
8.6
4
11.2 + 1.6
2
15.8
5
58.8 * 7.4
4
69.6 i 9.6
” Actinomycin (0.1 wg/ml) was injected at various times after the blood meal. The fat body was removed 18 hr PBM and incubated in medium containing actinomycin D (10 @g/ml) and phenylalanine-3H (50 pCi/ml) for 4 hr. The control tissues were incubated in medium without actinomycin. The data are from three independent experiments. Each number represents the mean of duplicate samples taken from replicates of 10 fat bodies each + the standard error when n > 2. b Post blood meal.
the total RNA of the fat body and its ability to synthesize vitellogenin begin to fall precipitously. We have also found that injection of actinomycin in uiuo at various times after the blood meal prevents the normal increase in vitellogenin synthesis, but allows synthesis to proceed at the rate expected for tissues assayed at the time of injection. This concept is presented diagrammatically in Fig. 6. When the kinetics of vitellogenin synthesis in the presence of actinomycin were examined, the rate of synthesis was constant for 6 hr. This implies that, once synthesized, the vitellogenin mRNA is relatively stable. With the aid of the above data the probable events occurring in the fat body after the blood meal can be reconstructed as follows: the blood meal triggers some un-
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31, 1973
known mechanism which results, 2 hr later, in the activation of RNA synthesis. Both ribosomal RNA and vitellogenin mRNA are synthesized, resulting in a tripling of the total RNA of the cell and rapid elaboration of protein synthetic machinery. The vitellogenin mRNA is stable, hence it accumulates, and beginning 3-4 hr after the blood meal the fat body begins to synthesize vitellogenins. The rate of vitellogenin synthesis rises until at 28 hr after the blood meal the RNA of the fat body is broken down and synthesis of vitellogenin declines. This reconstruction is speculative and is presented as a working hypothesis postulating transcriptional control of vitellogenin synthesis. How well do the present data agree with the hypothesis? A critical question relates to the kinds of RNA made during the increase in total RNA. We do not have direct evidence on this point but the results do suggest an answer.
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FIG. 6. The effect of in uiuo injection of actinomytin D on vitellogenin synthesis as measured in vitro. A diagrammatic representation of Fig. 2, redrawn to emphasize the early time points, and the data from Table 4. Arrows indicate time of actinomycin injection, -*time of assay, - - -, level of synthesis at 5 hours, level of synthesis at 12 hr.
HAGEDORN, FALLON, AND LAUFER
If we look first at Fig. 1 it is apparent that the total RNA of the fat body triples from 2 to 12 hr after feeding: this is almost certainly due primarily to accumulation of ribosomal RNA. If the ability to synthesize vitellogenin were limited by the availability of ribosomes, vitellogenin synthesis should also plateau at 12 hr. Figure 2, however, shows that vitellogenin synthesis continues to rise for another 16 hr. Thus it appears that during the first 12 hr both mRNA and the protein synthetic machinery are developed simultaneously. It is likely that after this period the increased rate of synthesis is due solely to continued synthesis and accumulation of vitellogenin mRNA. Several questions can be posed using the above model. One of these involves the nature of the “unknown mechanism” which triggers RNA synthesis. Hormones are certainly involved as the work of Clements (1956), Gillett (1956, 1957), Larsen (1958), Larsen and Bodenstein (1959), and Lea (1967) indicates. Recent work in the field of hormonal control of protein synthesis in other systems indicates that protein synthesis can be stimulated either without prior RNA synthesis or with an obligatory synthesis of RNA before protein synthesis begins (Schmike and Doyle, 1970; Lee et al., 1970). The hormonal control of vitellogenin synthesis in the mosquito is apparently of the latter type. Recent work in this laboratory has demonstrated that the ovary is the source of a humoral factor involved in activating the fat body (Hagedorn and Fallon, in preparation). Other questions one might pose to test the hypothesis of transcriptional control relate to identifying the kinds of RNA being made after the activation period and the nature of the mechanisms involved in the decline of vitellogenin synthesis after 28 hr. The unique aspects of this system which have been outlined here provide an unusual opportunity to study the hormonal
Vitellogenin
control tein.
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Thanks are due to James P. Calvet and Lee Weber for stimulating discussions during the course of this work. REFERENCES ANDERSON, E. (1964). Oocyte differentiation and vitellogenesis in the roach Periplaneta americana. J. Cell Biol. 20, 131-155. ANDERSON, L. M., and TELFER, W. H. (1970). Trypan blue inhibition of yolk deposition-a clue to follicle cell function in the cecropia moth. J. Embryol. Exp. Morphol. 23, 35-52. ANDERSON, W. A. and SPIELMAN, A. (1971). Permeability of the ovarian follicle of Aedes aegypti mosquitoes. J. Cell Biol. 50, 201-221. BELL, W. J. (1970). Demonstration and characterization of two vitellogenic blood proteins in Periplaneta americana: an immunochemical analysis. J. Insect Physiol. 16, 291-299. BROOKES, V. J. (1969). The induction of yolk protein synthesis in the fat body of an insect, Leucophaea maderae, by an analog of the juvenile hormone. Develop. Biol. 20, 459-471. CLEMENTS, A. N. (1956). Hormonal control of ovary development in mosquitoes. J. Exp. Biol. 33, 211223. ENGELMANN, F. (1969). Female specific protein: Biosynthesis controlled by corpus allatum in Leucophaea maderae. Science 165, 407-409. GILES, K. W. and MEYERS, A. (1965). An improved diphenylamine method for the estimation of deoxyribonucleic acid. Nature (London) 206, 93. GILLETT, J. D. (1956). Initiation and promotion of ovarian development in the mosquito Aedes aegypti. Ann. 7’rop. Med. Parasitol. 50, 375-380. GILI~ETT, J. D. (1957). Variation in the time of release of the ovarian development hormone in Aedes aegypti. Nature (London) 180, 656. GROSS, P. R. (1968). Actinomycin in Developmental Biology. In “Actinomycin, Nature, Formation and Activities” (S. A. Waksman, ed.), pp. 87-100. Wiley, New York. GROSS, P. R., and COLJSINEAU,G. H. (1964). Macromolecule synthesis and the influence of actinomytin on early development. Exp. Cell. Res. 33, 368395. GROSS, P. R., MALKIN, L. I., and MOYER, W. A. (1964). Templates for the first proteins of embryonic development. Proc. Nat. Acad. Sci. U.S. 51, 407-141. HAGEDORN, H. H., and JUDSON, C. L. (1972). Purification and site of synthesis of Aedes aegypti yolk proteins. J. Exp. 2001. 182, 367. HONIG, G. R., and RABINOVITZ, M. (1966). Inhibition by actinomycin D of oxidation dependent biosyn-
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