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Juvenile hormone-stimulated polyploidy in adult locust fat body
DEVELOPMENTAL
BIOLOGY
81,356-360
(1981)
Juvenile Hormone-Stimulated
Polyploidy
in Adult Locust
Fat Body
K. K. NAIR,* THOMAS T. CHEN,t AND G. R. WYATT* *Pestologg Centre, Department of Biological Sciences, Simon FraSeT University, Burnabg, British Columbia V5A lS6, tDepartment of Biology, Me&laster University, Hamilton, Ontario L8S 4K1, and $Groupin Eukaryotic hfOkCUhT Biology and Evolution, Department of Biology, Queen’s University, Kingston, Ontario K7L 8N6, Canada Received March 27, 1980; accepted in revised form June 13, 1980 The relative DNA contents of adult fat body nuclei of Locusta migratwia have been assayed by scanning microspectrophotometry after Feulgen staining, spermatid and brain nuclei being used as haploid and diploid reference standards. In the fat body, several DNA classes were found, the mean ploidy increasing during the period of reproductive maturation. The extinction frequency peaks fall at values that are less than doublings of the diploid value, which suggests incomplete replication of the genome, although this interpretation requires confirmation by independent techniques. On Day 1 the female and male fat body nuclei are chiefly tetraploids. Elevation to octaploidy or higher, especially in the female is evident by Day 15. Allatectomy, removing the source of JH, prevents the increase in ploidy, and administration of the JH mimic, ZR-515, restores it in both sexes. Incorporation of rH]thymidine in female fat body shows a broad peak, maximal at Day 6. After allatectomy, incorporation of [‘Hjthymidine is strongly stimulated by ZR-515 in both sexes. It is suggested that the JH-dependent polyploidy in female fat body permits accelerated production of mRNA for vitellogenin and possibly other proteins. The male fat body responds to JH by DNA replication but not by expression of the vitellogenin gene. MATERIALS
INTRODUCTION
Polyploidy occurs in many cell types that are differentiated for the production of large amounts of particular proteins, and is especially common in insects (Brodsky and Uryvaeva, 1977; Nagl, 1978). In our studies on the juvenile hormone (JH)-dependent synthesis of the yolk precursor protein, vitellogenin, in the fat body of the adult female migratory locust, we observed a marked increase in total tissue DNA close to the time of onset of vitellogenin production (Chen et al., 1979). The fat body also showed nuclear enlargement and increased basophilia, but no evidence of mitosis (Lauverjat, 1977; Couble et al., 1979). These observations raised the questions whether the increase in DNA represented enhanced ploidy, how it might be related to the action of JH and the synthesis of vitellogenin, and whether parallel events occur in the male fat body, which does not produce vitellogenin (Chen et al., 1979). The presence of several ploidy classes in the fat body and other tissues of locusts has previously been reported, but developmental changes were not studied (Fox, 1970). We have now examined the influences of age, withdrawal of JH, and administration of a JH analog on female and male fat body, using microspectrophotometry of Feulgen-stained preparations to detect several nuclear DNA classes and the incorporation of [3H]thymidine to assay for DNA synthesis. A preliminary report of our findings has already been given (Chen et al., 1977). 0012-1606/81/020356-05$02.00/O Copyright All rights
0 1981 by Academic Press. Inc. of reproduction in any form reserved.
AND METHODS
Locusta migratoria migratorioides originating from the Centre for Overseas Pest Research, London, was reared on a bran mixture and wheat seedlings under crowded conditions with 16-hr day length, as previously described (Chen et al., 1978, 1979). Females were kept in the presence of mature males from the time of emergence as adults (Couble et al., 1979). Allatectomy was performed by a modification of the method of Strong (1963). In one late experiment (Fig. 2, as noted), instead of allatectomy, the corpora allata were destroyed by the topical application, within 24 hr after eclosion, of 500 pg of a precocene analog, 7-ethoxy, 6-methoxy-2, 2-dimethyl chromene (Bowers, 1977), the gift of Dr. G. B. Staal, in acetone (Abu-Hakima and Wyatt, 1980). The juvenile hormone mimic, ZR-515 (methoprene; Henrick et al., 1973), kindly provided by Dr. G. B. Staal (Zoecon Corporation, Palo Alto, Calif. was applied topically at a dose of 200 pg in acetone. The allatectomized controls were treated with 50 ~1 of acetone. This hormone analog and dose were chosen on the basis of previously determined dose-response curves for the stimulation of vitellogenin synthesis (Chen et al., 1979). Cytochemical measurement of DNA. Fat body, testis, or brain was rinsed in a locust Ringer solution (Chen et aZ., 1978), fixed in Carnoy’s fixative on ice, and stored in 70% ethanol until use. For Feulgen staining, tissues were subjected to hydrolysis in 3.5 N HCl at 37°C for 356
357
BRIEF NOTES
20 min and bulk-stained in Schiff’s reagent (basic fuchsin, Harleco). The stained tissue was squashed on gelatinized slides and the cover slips were removed by the dry ice technique. After dehydration and clearing in xylene, the squashes were mounted in oil of matching refractive index 1.556 (Cargille, Cedar Grove, N. J.). Extinction measurements of Feulgen-stained nuclei were carried out at 570 nm with a scanning microscope photometer (SMP) (Carl Zeiss, West Germany). The diameter of the scanning aperture was 1.4 pm. The SMP was on line with a PDP-12 computer (Digital Equipment Corp.) which facilitated scanning at predetermined intervals of 1 pm. (For details of the instrumentation see Mahon and Nair, 1975.) From each group, consisting of two to three insects, a total of 100-400 nuclei were measured. For graphic presentation of the different ploidy levels, the total extinction for each nucleus was expressed as its log, value, since an increase of one unit on the log, scale represents a doubling of DNA content. Incorporation of [3H$hymidine. For measurement of DNA synthesis, the fat bodies of individual locusts were incubated in 1 ml of Ringer solution containing 4 PCi of [3H]thymidine (20 Ci/mmole; Amersham Corporation) at 30°C for 3 hr. The fat body was then homogenized in ethanol and the residue washed and counted as previously described for the incorporation of [3H]uridine (Chen et al., 1979). RESULTS
The DNA Classes of Fat Body Nuclei
DNA CONTENT
( LOG2 TOTAL EXTINCTION)
FIG. 1. Frequency distribution of DNA classes in nuclei of spermatids, brain, and fat body of adult L. migratoria, determined by Feulgen staining and microspectrophotometric scanning. Numbers of nuclei measured for each tissue are given in parentheses. (A), Spermatids (100); (B), brain (50 female, 100 male); (C-G), fat body; (C), Day 1 female (200); (D), Day 8 female (400); (E), Day 15 female (200); (F), Day 1 male (100); (G), Day 15 male (100).
To provide reference values for interpretation of the Feulgen extinction readings from fat body nuclei, a series of spermatid and female and male adult brain nu- would represent successive levels of polyploidy. In the clei were measured (Figs. lA, B). The distributions were l-day-old female the major peak falls between the preunimodal, with means of 16.2 f 1.3 (SD) (n = 100) dicted 2C and 4C, while the minor peak falls between Z&, nmunits for the spermatids, and 32.6 + 1.7 (n = 50) 4C and 8C values. Since the distance between these two and 33.7 + 2.6 (n = 100) for female and male brains, peaks is one unit apart on the log2 scale it is assumed respectively. From the average of these, the 2C value that the Day 1 pattern for the females represents tetis taken as 32.9 ,?&, nm.This corresponds to 12.7 pg of raploids plus a few octaploids, whereas on Day 8 the DNA, which has been estimated as the diploid comple- nuclei are chiefly octaploids. Although on Day 15 the peak falls at 8C we cannot state equivocally that these ment for L. migratoria (Rees et al., 1978). Data on fat body from female locusts 1,8, and 15 days nuclei are actually octaploids or 16 ploids. Male fat body and from males 1 and 15 days after adult eclosion are is also predominantly tetraploid on Day 1, but undershown in Fig. 1. Fat body of each age shows two or more goes less replication than the female tissue, to become classes of nuclei with respect to DNA content, the dis- octaploid by Day 15. After allatectomy of locusts of either sex, the nuclear tribution shifting toward higher values with increasing age. The observed peaks do not coincide precisely with DNA distribution remained almost unchanged from the successive doublings of the diploid complement esti- Day 1 pattern (Fig. 2). Five days after treatment of mated as above. It is not certain whether this is due to allatectomized locusts with ZR-515, the pattern had lack of stoichiometry in the Feulgen staining of these shifted to the right, indicating one and two cycles of nuclei or whether it reflects incomplete replication of DNA replication induced by the hormone mimic in the genome (see Discussion), but, in either case, peaks males and females, respectively.
358
DEVELOPMENTALBIOLOGY
Incorporation
VOLUME 81, 1981
of [3HJThymidine
As an indicator of DNA synthesis, we measured the incorporation of [3H]thymidine into the DNA of fat body from female locusts of various ages during 3 hr incubation in vitro (Fig. 3A). The incorporation values from individual locusts of the same age varied greatly and were not normally distributed, which suggests differences in the timing of maturation. The data do, however, show a broad peak of synthesis, maximal at Day 6 (144 hr) and extending roughly from Day 5 to Day 9 and later in some individuals. After administration of ZR-515 to allatectomized locusts there was strong stimulation of thymidine incorporation. In a series of females (Fig. 3B), incorporation was observed in some individuals by 24 hr and in all at 36 and 48 hr, and declined gradually thereafter. Again, there was great variation among the population. Because of poor survival after allatectomy, only a small number of males were tested for the effect of ZR-515; the data obtained, however (Fig. 3C), are sufficient to confirm the stimulation of DNA synthesis by the hormone in males, and to suggest that the effect may be more rapid than in females. DISCUSSION
An increase in total fat body DNA during the maturation of adult female Locusta has already been reported, being steepest at about Day 6 after eclosion and amounting to an approximate doubling by Day 15 (Chen
5
I B
4 3 2 10 0
1 5
6
7
8
9
5
6
7
8
DNA CONTENT (LOG2 TOTAL EXTINCTION)
FIG. 2. Effects of allatectomy and treatment with ZR-515 on polyploidization in adult fat body. Locusts were allatectomized or treated with precocene within 1 day after adult emergence and used 14-20 days later. The JH analog, ZR-515, was applied as 200 pg topically 5 days before dissection. (A), Allatectomized female (400); (B), same treated with ZR-515 (400); (C), precocene-treated male (100); (D), same treated with ZR-515 (100).
~:;:l;;r:l-
8 . I/ a
k
100 HOURS AFTER E&IS
300 H;URS
A:?ER
Zi%5
TFi:iTMEdy
.
k ‘2 B
op.
. .
0
:
:
;
l
0
.
.
. 1
0
FIG. 3. Incorporation of t3H]thymidine into DNA of adult fat body. Incorporation took place during incubation of fat body in vitro for 3 hr and samples were prepared for counting as described under Materials and Methods. (A), Normally developing adult females; (B), allatectomized females treated with ZR-515 as in Fig. 2 and taken at different times; (C), allatectomized males treated with ZR-515 in the same manner. (a), Values from individual locust fat bodies; (o), means for each age or time group.
et al., 1979). In subsequent analyses of male tissue (unpublished) we found a similar but somewhat smaller increase in DNA during maturation. The results of a more extended series of analyses on both sexes (Irvine, 19’79) indicate a somewhat more complex picture, with continued increase in the average fat body DNA content up to Day 24 after emergence and marked divergence in the developmental rates of individuals in the population. This can account for some of the variability among individuals of any given age. No mitosis has been observed in adult locust fat body (Lauverjat, 1977; Couble et al., 19’79), and we have now shown that the DNA replication leads to increased polyploidy. This conforms to a pattern observed in a number of differentiated animal and plant tissues, particularly where there is specialization for massive synthesis of one or a few proteins (Brodsky and Uryvaeva, 1977; Nagl, 1978). The observed frequency distribution peaks for the DNA levels in the polyploid nuclear classes, however, fall distinctly below the predicted values for successive doublings of the diploid amount. In an earlier study of Locusta tissue nuclei by a similar technique, Fox (1970) also noted an apparent less than doubling between DNA classes, and suggested that this represented underreplication, a portion of the genome being omitted from replication at each cycle, as part of the mechanism of tissue differentiation. In a current study on another
359
BRIEF NOTES
locust, Schistocerca gregaria, Kooman and Nair (unpublished) have also observed that the DNA classes in the polyploid fat body nuclei were not exact multiples of the 2C values from brain nuclei. Nag1 (1978) has reviewed a number of papers relating to underreplication and selective amplification of DNA in animal and plant cells. In the polytene chromosomes of larval Drosophila it is firmly established that the rRNA genes are underreplicated and some heterochromatic regions are replicated little if at all (Gall et al., 1971). Tissue-specific differential replication of several satellite components in the DNA of polyploid tissues of adult Drosophila has also been demonstrated (Endow and Gall, 1975). Underreplication of heterochromatin has been shown in several other Diptera, but, among nondipterous insects, the only clear examples appear to be in certain Homoptera, in which heterochromatin is segregated in distinct chromosome sets (Lorick, 1970). In certain plants, satellite DNA forms a lower percentage of the total in differentiated than in meristematic cells (e.g., Cucumis; Pearson et al., 1974). In the fibroin-secreting cells of the silkworm silk gland, on the other hand, which exhibit the highest levels of polyploidy known, no evidence for selective DNA replication could be found (Gage, 1974). The possibility of partial replication in locusts is therefore of considerable interest. Cytophotometric data alone cannot provide decisive evidence, since the intensity of Feulgen staining can be influenced by the degree of condensation of the chromatin (Lorick, 1970), and some intermediate values may be attributed to nuclei in the S phase (Roberts and Roberts, 1972). Irvine (1979) has shown an apparently doubling series of nuclear volumes in adult Locusta fat body. Biochemical studies on the DNA of locust tissues in order to resolve the question of partial replication are in progress. DNA synthesis and the increase in polyploidy in the adult locust fat body clearly depend on the action of JH, as shown by the effects of allatectomy and replacement by the JH mimic, ZR-515. Increased accumulation of DNA in the silkworm silk gland after treatment with ZR-515 has been reported, but this occurred only under suboptimal growth conditions and was associated with prolongation of the larval instar (Shigematsu et al., 1978). In the cockroach, Periplaneta americana, JHdependent vitellogenesis is accompanied by DNA synthesis in the follicle cells, but the DNA synthesis itself apparently does not require JH (Bell and Sams, 1974). In another cockroach, Leucophaea maderae, however, JH-dependent ovarian DNA synthesis has recently been reported (Wellman and Koeppe, 1978). We have now shown that in locusts DNA synthesis can be a major, early event in the action of JH on a target tissue. In
normal adult female maturation, maximal DNA synthesis is observed on Day 6, which precedes the major rise in RNA and vitellogenin synthesis that starts about Day 8 (Chen et al., 1979). After experimental application of ZR-515, also, the rise in DNA synthesis precedes that in vitellogenin synthesis. The sequential and possibly causal relationships between DNA replication and the production of various classes of RNA remain to be established. Polyploidy in the adult female fat body presumably serves to provide multiple copies of the genome from which the vitellogenin (or other) genes can be selectively expressed to provide the required high levels of specific messenger RNA. In an analogous system, the estrogen-induced synthesis of vitellogenin in amphibian liver is preceded by replication of DNA, but this appears not to be essential for the induction of the vitellogenin genes (Tata, 1978). That the DNA synthesis in the locust fat body may not be directly linked with turning on the vitellogenin genes is suggested by the fact that JH-dependent polyploidy develops also in male fat body, in which these genes remain inactive. Thus, the male fat body is competent to respond to JH by DNA replication but differs from the female tissue in its programming for the synthesis of specific proteins. This sex-specific programming of hormonal inducibility must depend upon developmental events that precede the emergence of the adult insect. We thank Dr. P. Couble for help in the initial stages of this study, Mr. J. Irvine for discussion and sharing of results, Cathy Kooman and Jann Aitken for assistance in microspectrophotometry. This work was supported by grants from the Natural Sciences and Engineering CouncilofCanadatoK.K.N.,T.T.C.,andG.R.W.andagranttoG.R.W. from the U. S. National Institutes of Health (HD-0’7951). REFERENCES ABU-HAKIMA, R., and WYATT, G. R. (1980). Vitellogenin synthesis induced in locust fat body by juvenile hormone added in vitro. Submitted for publication. BELL, W. J., and SAMS, G. R. (1974). Factors promoting vitellogenic competence and yolk deposition in the cockroach ovary: The postecdysis female. J. Insect Physiol. 20, 2475-2485. BOWERS, W. S. (1977). Anti-Juvenile hormones from plants: Chemistry and biological activity. P&f Acad. Scient. Scripta Varia 42, 129-156. BRODSKY, W. Y., and URYVAEVA, I. V. (1977). Cell polyploidy: Its relation to tissue growth and function. Int. Rev. Cytol. 50,275-332. CHEN, T. T., COUBLE, P., NAIR, K. K., and WYATP, G. R. (1977). Juvenile hormone-induced DNA synthesis in the adult locust fat body. Canad. Fed. Biol. Sci. Proc. 20, 181. CHEN, T. T., STRAHLENDORF, P. W., and WYATT, G. R. (1978). Vitellin and vitellogenin from locusts (Locusta migratoria). Properties and post-translational modification in the fat body. J. Biol. Chem. 253, 5325-5331. CHEN, T. T., COUBLE, P., ABU-HAKIMA, R., and WYATT, G. R. (1979). Juvenile hormone-controlled vitellogenin synthesis in Locusta mi-
360
DEVELOPMENTALBIOLOGY
gnitwria fat body. Hormonal induction in wivo. Develop. BioL 69, 59-72. COUBLE,P., CHEN, T. T., and WYATT, G. R. (1979). Juvenile hormonecontrolled vitellogenin synthesis in Locusta migratoria fat body: Cytological development. J. Insect Physi~l. 25,327-337. ENDOW,S. A., and GALL, J. G. (1975). Differential replication of satellite DNA in polyploid tissues of Drosophila vi&s. Chrom.osoma 50.175-192. FOX, D. P. (1970). A non-doubling DNA series in somatic tissues of the locusts Schistocerca gregaria and Locusta migratoria. Chromosoma 29,448-461. GAGE, L. P. (1974). Polyploidization of the silk gland of Bombyx mori. J. Mol. Biol. 86,97-108.
GALL,,J. G., COHEN,E. H., and POLAN, M. L. (1971). Repetitive DNA sequences in Drosophila. Chromosoma 33, 319-344. HENRICK, C. A., STAAL, G. B., and SIDDALL, J. B. (1973). Alkyl3,7,11trimethyl-2,4-dodecadienoate, a new class of potent insect growth regulators with juvenile hormone activity. J. Agr. Food Chem. 21, 354-35s. IRVINE, D. J. (1979). “The Development of Polyploidy in the Fat Body of Locwta migratoria.” M.Sc. thesis, Queen’s University, Kingston, Ontario. LAUVERJAT, S. (1977). L’evolution post-imaginal du tissue adipeux femelle de Locusta migratoria et son controle endocrine. Gen. Camp. EndocrinoL
33,13-34.
LORICK,G. (1970). Differential DNA synthesis in heterochromatic and
VOLUME81, 1981
euchromatic chromosome sets of Planoccus citri. Chromosoma 32, 11-30. MAHON, D. C., and NAIR, K. K. (1975). Stoichiometry of the aldehyde fuchsin staining reaction for proteins. J. Histochem. Cytochem. 23, 652-659. NAGL, W. (1978). “Endopolyploidy and Polyteny in Differentiation and Evolution.” North-Holland, Amsterdam. PEARSON,G. G., TIMMIS, J. N., and INGLE, J. (1974). The differential replication of DNA during plant development. Chromoxnna 45, 281-294. REES,H., SHAW,D. D., and WILKINSON,P. (1978). Nuclear DNA variation among acridid grasshoppers. Proc. R. Sot. London Ser. B 202, 517-525. ROBERTS,B., and ROBERTS,S. (1972). The DNA content of testes and malpighian tubule nuclei of Tricholiqproctia impatient (Sarcophagidae, Diptera). Chromosoma 39,83-94. SHIGEMATSU,H., KURATA, K., and TAKESHITA, H. (1978). Nucleic acids accumulation of silk gland of Bombyx mori in relation to silk protein. Camp. Physiol. Biochem. 61B. 237-242. STRONG,L. (1963). A simple apparatus for use in removing the corpora allata from locusts. Bull. Entomol. Res. 54, 19-21. TATA, J. R. (1978). Induction and regulation of vitellogenin synthesis by estrogen. In “Biochemical Actions of Hormones” (G. Litwack, ed.), Vol. 5, pp. 397-431. Academic Press, New York. WELLMAN, S. C., and KOEPPE, J. K. (1978). Ovarian maturation in Leucophaea maderae: Ovarian growth pattern and induction of ovarian DNA synthesis with juvenile hormone. Amer. 2001. 18,637.
BIOLOGY
81,356-360
(1981)
Juvenile Hormone-Stimulated
Polyploidy
in Adult Locust
Fat Body
K. K. NAIR,* THOMAS T. CHEN,t AND G. R. WYATT* *Pestologg Centre, Department of Biological Sciences, Simon FraSeT University, Burnabg, British Columbia V5A lS6, tDepartment of Biology, Me&laster University, Hamilton, Ontario L8S 4K1, and $Groupin Eukaryotic hfOkCUhT Biology and Evolution, Department of Biology, Queen’s University, Kingston, Ontario K7L 8N6, Canada Received March 27, 1980; accepted in revised form June 13, 1980 The relative DNA contents of adult fat body nuclei of Locusta migratwia have been assayed by scanning microspectrophotometry after Feulgen staining, spermatid and brain nuclei being used as haploid and diploid reference standards. In the fat body, several DNA classes were found, the mean ploidy increasing during the period of reproductive maturation. The extinction frequency peaks fall at values that are less than doublings of the diploid value, which suggests incomplete replication of the genome, although this interpretation requires confirmation by independent techniques. On Day 1 the female and male fat body nuclei are chiefly tetraploids. Elevation to octaploidy or higher, especially in the female is evident by Day 15. Allatectomy, removing the source of JH, prevents the increase in ploidy, and administration of the JH mimic, ZR-515, restores it in both sexes. Incorporation of rH]thymidine in female fat body shows a broad peak, maximal at Day 6. After allatectomy, incorporation of [‘Hjthymidine is strongly stimulated by ZR-515 in both sexes. It is suggested that the JH-dependent polyploidy in female fat body permits accelerated production of mRNA for vitellogenin and possibly other proteins. The male fat body responds to JH by DNA replication but not by expression of the vitellogenin gene. MATERIALS
INTRODUCTION
Polyploidy occurs in many cell types that are differentiated for the production of large amounts of particular proteins, and is especially common in insects (Brodsky and Uryvaeva, 1977; Nagl, 1978). In our studies on the juvenile hormone (JH)-dependent synthesis of the yolk precursor protein, vitellogenin, in the fat body of the adult female migratory locust, we observed a marked increase in total tissue DNA close to the time of onset of vitellogenin production (Chen et al., 1979). The fat body also showed nuclear enlargement and increased basophilia, but no evidence of mitosis (Lauverjat, 1977; Couble et al., 1979). These observations raised the questions whether the increase in DNA represented enhanced ploidy, how it might be related to the action of JH and the synthesis of vitellogenin, and whether parallel events occur in the male fat body, which does not produce vitellogenin (Chen et al., 1979). The presence of several ploidy classes in the fat body and other tissues of locusts has previously been reported, but developmental changes were not studied (Fox, 1970). We have now examined the influences of age, withdrawal of JH, and administration of a JH analog on female and male fat body, using microspectrophotometry of Feulgen-stained preparations to detect several nuclear DNA classes and the incorporation of [3H]thymidine to assay for DNA synthesis. A preliminary report of our findings has already been given (Chen et al., 1977). 0012-1606/81/020356-05$02.00/O Copyright All rights
0 1981 by Academic Press. Inc. of reproduction in any form reserved.
AND METHODS
Locusta migratoria migratorioides originating from the Centre for Overseas Pest Research, London, was reared on a bran mixture and wheat seedlings under crowded conditions with 16-hr day length, as previously described (Chen et al., 1978, 1979). Females were kept in the presence of mature males from the time of emergence as adults (Couble et al., 1979). Allatectomy was performed by a modification of the method of Strong (1963). In one late experiment (Fig. 2, as noted), instead of allatectomy, the corpora allata were destroyed by the topical application, within 24 hr after eclosion, of 500 pg of a precocene analog, 7-ethoxy, 6-methoxy-2, 2-dimethyl chromene (Bowers, 1977), the gift of Dr. G. B. Staal, in acetone (Abu-Hakima and Wyatt, 1980). The juvenile hormone mimic, ZR-515 (methoprene; Henrick et al., 1973), kindly provided by Dr. G. B. Staal (Zoecon Corporation, Palo Alto, Calif. was applied topically at a dose of 200 pg in acetone. The allatectomized controls were treated with 50 ~1 of acetone. This hormone analog and dose were chosen on the basis of previously determined dose-response curves for the stimulation of vitellogenin synthesis (Chen et al., 1979). Cytochemical measurement of DNA. Fat body, testis, or brain was rinsed in a locust Ringer solution (Chen et aZ., 1978), fixed in Carnoy’s fixative on ice, and stored in 70% ethanol until use. For Feulgen staining, tissues were subjected to hydrolysis in 3.5 N HCl at 37°C for 356
357
BRIEF NOTES
20 min and bulk-stained in Schiff’s reagent (basic fuchsin, Harleco). The stained tissue was squashed on gelatinized slides and the cover slips were removed by the dry ice technique. After dehydration and clearing in xylene, the squashes were mounted in oil of matching refractive index 1.556 (Cargille, Cedar Grove, N. J.). Extinction measurements of Feulgen-stained nuclei were carried out at 570 nm with a scanning microscope photometer (SMP) (Carl Zeiss, West Germany). The diameter of the scanning aperture was 1.4 pm. The SMP was on line with a PDP-12 computer (Digital Equipment Corp.) which facilitated scanning at predetermined intervals of 1 pm. (For details of the instrumentation see Mahon and Nair, 1975.) From each group, consisting of two to three insects, a total of 100-400 nuclei were measured. For graphic presentation of the different ploidy levels, the total extinction for each nucleus was expressed as its log, value, since an increase of one unit on the log, scale represents a doubling of DNA content. Incorporation of [3H$hymidine. For measurement of DNA synthesis, the fat bodies of individual locusts were incubated in 1 ml of Ringer solution containing 4 PCi of [3H]thymidine (20 Ci/mmole; Amersham Corporation) at 30°C for 3 hr. The fat body was then homogenized in ethanol and the residue washed and counted as previously described for the incorporation of [3H]uridine (Chen et al., 1979). RESULTS
The DNA Classes of Fat Body Nuclei
DNA CONTENT
( LOG2 TOTAL EXTINCTION)
FIG. 1. Frequency distribution of DNA classes in nuclei of spermatids, brain, and fat body of adult L. migratoria, determined by Feulgen staining and microspectrophotometric scanning. Numbers of nuclei measured for each tissue are given in parentheses. (A), Spermatids (100); (B), brain (50 female, 100 male); (C-G), fat body; (C), Day 1 female (200); (D), Day 8 female (400); (E), Day 15 female (200); (F), Day 1 male (100); (G), Day 15 male (100).
To provide reference values for interpretation of the Feulgen extinction readings from fat body nuclei, a series of spermatid and female and male adult brain nu- would represent successive levels of polyploidy. In the clei were measured (Figs. lA, B). The distributions were l-day-old female the major peak falls between the preunimodal, with means of 16.2 f 1.3 (SD) (n = 100) dicted 2C and 4C, while the minor peak falls between Z&, nmunits for the spermatids, and 32.6 + 1.7 (n = 50) 4C and 8C values. Since the distance between these two and 33.7 + 2.6 (n = 100) for female and male brains, peaks is one unit apart on the log2 scale it is assumed respectively. From the average of these, the 2C value that the Day 1 pattern for the females represents tetis taken as 32.9 ,?&, nm.This corresponds to 12.7 pg of raploids plus a few octaploids, whereas on Day 8 the DNA, which has been estimated as the diploid comple- nuclei are chiefly octaploids. Although on Day 15 the peak falls at 8C we cannot state equivocally that these ment for L. migratoria (Rees et al., 1978). Data on fat body from female locusts 1,8, and 15 days nuclei are actually octaploids or 16 ploids. Male fat body and from males 1 and 15 days after adult eclosion are is also predominantly tetraploid on Day 1, but undershown in Fig. 1. Fat body of each age shows two or more goes less replication than the female tissue, to become classes of nuclei with respect to DNA content, the dis- octaploid by Day 15. After allatectomy of locusts of either sex, the nuclear tribution shifting toward higher values with increasing age. The observed peaks do not coincide precisely with DNA distribution remained almost unchanged from the successive doublings of the diploid complement esti- Day 1 pattern (Fig. 2). Five days after treatment of mated as above. It is not certain whether this is due to allatectomized locusts with ZR-515, the pattern had lack of stoichiometry in the Feulgen staining of these shifted to the right, indicating one and two cycles of nuclei or whether it reflects incomplete replication of DNA replication induced by the hormone mimic in the genome (see Discussion), but, in either case, peaks males and females, respectively.
358
DEVELOPMENTALBIOLOGY
Incorporation
VOLUME 81, 1981
of [3HJThymidine
As an indicator of DNA synthesis, we measured the incorporation of [3H]thymidine into the DNA of fat body from female locusts of various ages during 3 hr incubation in vitro (Fig. 3A). The incorporation values from individual locusts of the same age varied greatly and were not normally distributed, which suggests differences in the timing of maturation. The data do, however, show a broad peak of synthesis, maximal at Day 6 (144 hr) and extending roughly from Day 5 to Day 9 and later in some individuals. After administration of ZR-515 to allatectomized locusts there was strong stimulation of thymidine incorporation. In a series of females (Fig. 3B), incorporation was observed in some individuals by 24 hr and in all at 36 and 48 hr, and declined gradually thereafter. Again, there was great variation among the population. Because of poor survival after allatectomy, only a small number of males were tested for the effect of ZR-515; the data obtained, however (Fig. 3C), are sufficient to confirm the stimulation of DNA synthesis by the hormone in males, and to suggest that the effect may be more rapid than in females. DISCUSSION
An increase in total fat body DNA during the maturation of adult female Locusta has already been reported, being steepest at about Day 6 after eclosion and amounting to an approximate doubling by Day 15 (Chen
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4 3 2 10 0
1 5
6
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6
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8
DNA CONTENT (LOG2 TOTAL EXTINCTION)
FIG. 2. Effects of allatectomy and treatment with ZR-515 on polyploidization in adult fat body. Locusts were allatectomized or treated with precocene within 1 day after adult emergence and used 14-20 days later. The JH analog, ZR-515, was applied as 200 pg topically 5 days before dissection. (A), Allatectomized female (400); (B), same treated with ZR-515 (400); (C), precocene-treated male (100); (D), same treated with ZR-515 (100).
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FIG. 3. Incorporation of t3H]thymidine into DNA of adult fat body. Incorporation took place during incubation of fat body in vitro for 3 hr and samples were prepared for counting as described under Materials and Methods. (A), Normally developing adult females; (B), allatectomized females treated with ZR-515 as in Fig. 2 and taken at different times; (C), allatectomized males treated with ZR-515 in the same manner. (a), Values from individual locust fat bodies; (o), means for each age or time group.
et al., 1979). In subsequent analyses of male tissue (unpublished) we found a similar but somewhat smaller increase in DNA during maturation. The results of a more extended series of analyses on both sexes (Irvine, 19’79) indicate a somewhat more complex picture, with continued increase in the average fat body DNA content up to Day 24 after emergence and marked divergence in the developmental rates of individuals in the population. This can account for some of the variability among individuals of any given age. No mitosis has been observed in adult locust fat body (Lauverjat, 1977; Couble et al., 19’79), and we have now shown that the DNA replication leads to increased polyploidy. This conforms to a pattern observed in a number of differentiated animal and plant tissues, particularly where there is specialization for massive synthesis of one or a few proteins (Brodsky and Uryvaeva, 1977; Nagl, 1978). The observed frequency distribution peaks for the DNA levels in the polyploid nuclear classes, however, fall distinctly below the predicted values for successive doublings of the diploid amount. In an earlier study of Locusta tissue nuclei by a similar technique, Fox (1970) also noted an apparent less than doubling between DNA classes, and suggested that this represented underreplication, a portion of the genome being omitted from replication at each cycle, as part of the mechanism of tissue differentiation. In a current study on another
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BRIEF NOTES
locust, Schistocerca gregaria, Kooman and Nair (unpublished) have also observed that the DNA classes in the polyploid fat body nuclei were not exact multiples of the 2C values from brain nuclei. Nag1 (1978) has reviewed a number of papers relating to underreplication and selective amplification of DNA in animal and plant cells. In the polytene chromosomes of larval Drosophila it is firmly established that the rRNA genes are underreplicated and some heterochromatic regions are replicated little if at all (Gall et al., 1971). Tissue-specific differential replication of several satellite components in the DNA of polyploid tissues of adult Drosophila has also been demonstrated (Endow and Gall, 1975). Underreplication of heterochromatin has been shown in several other Diptera, but, among nondipterous insects, the only clear examples appear to be in certain Homoptera, in which heterochromatin is segregated in distinct chromosome sets (Lorick, 1970). In certain plants, satellite DNA forms a lower percentage of the total in differentiated than in meristematic cells (e.g., Cucumis; Pearson et al., 1974). In the fibroin-secreting cells of the silkworm silk gland, on the other hand, which exhibit the highest levels of polyploidy known, no evidence for selective DNA replication could be found (Gage, 1974). The possibility of partial replication in locusts is therefore of considerable interest. Cytophotometric data alone cannot provide decisive evidence, since the intensity of Feulgen staining can be influenced by the degree of condensation of the chromatin (Lorick, 1970), and some intermediate values may be attributed to nuclei in the S phase (Roberts and Roberts, 1972). Irvine (1979) has shown an apparently doubling series of nuclear volumes in adult Locusta fat body. Biochemical studies on the DNA of locust tissues in order to resolve the question of partial replication are in progress. DNA synthesis and the increase in polyploidy in the adult locust fat body clearly depend on the action of JH, as shown by the effects of allatectomy and replacement by the JH mimic, ZR-515. Increased accumulation of DNA in the silkworm silk gland after treatment with ZR-515 has been reported, but this occurred only under suboptimal growth conditions and was associated with prolongation of the larval instar (Shigematsu et al., 1978). In the cockroach, Periplaneta americana, JHdependent vitellogenesis is accompanied by DNA synthesis in the follicle cells, but the DNA synthesis itself apparently does not require JH (Bell and Sams, 1974). In another cockroach, Leucophaea maderae, however, JH-dependent ovarian DNA synthesis has recently been reported (Wellman and Koeppe, 1978). We have now shown that in locusts DNA synthesis can be a major, early event in the action of JH on a target tissue. In
normal adult female maturation, maximal DNA synthesis is observed on Day 6, which precedes the major rise in RNA and vitellogenin synthesis that starts about Day 8 (Chen et al., 1979). After experimental application of ZR-515, also, the rise in DNA synthesis precedes that in vitellogenin synthesis. The sequential and possibly causal relationships between DNA replication and the production of various classes of RNA remain to be established. Polyploidy in the adult female fat body presumably serves to provide multiple copies of the genome from which the vitellogenin (or other) genes can be selectively expressed to provide the required high levels of specific messenger RNA. In an analogous system, the estrogen-induced synthesis of vitellogenin in amphibian liver is preceded by replication of DNA, but this appears not to be essential for the induction of the vitellogenin genes (Tata, 1978). That the DNA synthesis in the locust fat body may not be directly linked with turning on the vitellogenin genes is suggested by the fact that JH-dependent polyploidy develops also in male fat body, in which these genes remain inactive. Thus, the male fat body is competent to respond to JH by DNA replication but differs from the female tissue in its programming for the synthesis of specific proteins. This sex-specific programming of hormonal inducibility must depend upon developmental events that precede the emergence of the adult insect. We thank Dr. P. Couble for help in the initial stages of this study, Mr. J. Irvine for discussion and sharing of results, Cathy Kooman and Jann Aitken for assistance in microspectrophotometry. This work was supported by grants from the Natural Sciences and Engineering CouncilofCanadatoK.K.N.,T.T.C.,andG.R.W.andagranttoG.R.W. from the U. S. National Institutes of Health (HD-0’7951). REFERENCES ABU-HAKIMA, R., and WYATT, G. R. (1980). Vitellogenin synthesis induced in locust fat body by juvenile hormone added in vitro. Submitted for publication. BELL, W. J., and SAMS, G. R. (1974). Factors promoting vitellogenic competence and yolk deposition in the cockroach ovary: The postecdysis female. J. Insect Physiol. 20, 2475-2485. BOWERS, W. S. (1977). Anti-Juvenile hormones from plants: Chemistry and biological activity. P&f Acad. Scient. Scripta Varia 42, 129-156. BRODSKY, W. Y., and URYVAEVA, I. V. (1977). Cell polyploidy: Its relation to tissue growth and function. Int. Rev. Cytol. 50,275-332. CHEN, T. T., COUBLE, P., NAIR, K. K., and WYATP, G. R. (1977). Juvenile hormone-induced DNA synthesis in the adult locust fat body. Canad. Fed. Biol. Sci. Proc. 20, 181. CHEN, T. T., STRAHLENDORF, P. W., and WYATT, G. R. (1978). Vitellin and vitellogenin from locusts (Locusta migratoria). Properties and post-translational modification in the fat body. J. Biol. Chem. 253, 5325-5331. CHEN, T. T., COUBLE, P., ABU-HAKIMA, R., and WYATT, G. R. (1979). Juvenile hormone-controlled vitellogenin synthesis in Locusta mi-
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gnitwria fat body. Hormonal induction in wivo. Develop. BioL 69, 59-72. COUBLE,P., CHEN, T. T., and WYATT, G. R. (1979). Juvenile hormonecontrolled vitellogenin synthesis in Locusta migratoria fat body: Cytological development. J. Insect Physi~l. 25,327-337. ENDOW,S. A., and GALL, J. G. (1975). Differential replication of satellite DNA in polyploid tissues of Drosophila vi&s. Chrom.osoma 50.175-192. FOX, D. P. (1970). A non-doubling DNA series in somatic tissues of the locusts Schistocerca gregaria and Locusta migratoria. Chromosoma 29,448-461. GAGE, L. P. (1974). Polyploidization of the silk gland of Bombyx mori. J. Mol. Biol. 86,97-108.
GALL,,J. G., COHEN,E. H., and POLAN, M. L. (1971). Repetitive DNA sequences in Drosophila. Chromosoma 33, 319-344. HENRICK, C. A., STAAL, G. B., and SIDDALL, J. B. (1973). Alkyl3,7,11trimethyl-2,4-dodecadienoate, a new class of potent insect growth regulators with juvenile hormone activity. J. Agr. Food Chem. 21, 354-35s. IRVINE, D. J. (1979). “The Development of Polyploidy in the Fat Body of Locwta migratoria.” M.Sc. thesis, Queen’s University, Kingston, Ontario. LAUVERJAT, S. (1977). L’evolution post-imaginal du tissue adipeux femelle de Locusta migratoria et son controle endocrine. Gen. Camp. EndocrinoL
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LORICK,G. (1970). Differential DNA synthesis in heterochromatic and
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euchromatic chromosome sets of Planoccus citri. Chromosoma 32, 11-30. MAHON, D. C., and NAIR, K. K. (1975). Stoichiometry of the aldehyde fuchsin staining reaction for proteins. J. Histochem. Cytochem. 23, 652-659. NAGL, W. (1978). “Endopolyploidy and Polyteny in Differentiation and Evolution.” North-Holland, Amsterdam. PEARSON,G. G., TIMMIS, J. N., and INGLE, J. (1974). The differential replication of DNA during plant development. Chromoxnna 45, 281-294. REES,H., SHAW,D. D., and WILKINSON,P. (1978). Nuclear DNA variation among acridid grasshoppers. Proc. R. Sot. London Ser. B 202, 517-525. ROBERTS,B., and ROBERTS,S. (1972). The DNA content of testes and malpighian tubule nuclei of Tricholiqproctia impatient (Sarcophagidae, Diptera). Chromosoma 39,83-94. SHIGEMATSU,H., KURATA, K., and TAKESHITA, H. (1978). Nucleic acids accumulation of silk gland of Bombyx mori in relation to silk protein. Camp. Physiol. Biochem. 61B. 237-242. STRONG,L. (1963). A simple apparatus for use in removing the corpora allata from locusts. Bull. Entomol. Res. 54, 19-21. TATA, J. R. (1978). Induction and regulation of vitellogenin synthesis by estrogen. In “Biochemical Actions of Hormones” (G. Litwack, ed.), Vol. 5, pp. 397-431. Academic Press, New York. WELLMAN, S. C., and KOEPPE, J. K. (1978). Ovarian maturation in Leucophaea maderae: Ovarian growth pattern and induction of ovarian DNA synthesis with juvenile hormone. Amer. 2001. 18,637.