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Synthesis of vitellogenic and non-vitellogenic yolk proteins by the fat body and the ovary of Leptinotarsa decemlineata
Comp. Biochem. Physiol. Vol. 83B, No. 1, pp. 251-254, 1986 Printed in Great Britain
0305-0491/86 $3.00+0.00 © 1986 Pergamon Press Ltd
SYNTHESIS OF VITELLOGENIC A N D N O N - V I T E L L O G E N I C YOLK PROTEINS BY THE F A T B O D Y A N D THE O V A R Y OF LEPTINOTARSA DECEMLINEATA M. PEFEROEN and A. DE LOOF Catholic University of Leuven, Zoological Institute, Naamsestraat 59, 3000 Leuven, Belgium (Tel: 016-22-2319) (Received 28 June 1985)
Abstract--1. Isolated ovaries of egg laying females synthesize and secrete three yolk proteins (two vitellogenins and chromoprotein 2). 2. The contribution of ovarian tissue to total yolk protein production is very small, the major site of synthesis of the three yolk proteins being the fat body. 3. There is a time lag between yolk protein synthesis by the fat body and yolk protein sequestration by the ovary. 4. In egg laying females, within 1 hr after the synthesis of both vitellogenins by the fat body, they appear in the oocytes as vitellins.
INTRODUCTION Sexually mature female insects synthesize some particular proteins which circulate in the haemolymph and are selectively accumulated by the oocytes. Until the late seventies, it was generally assumed that the fat body was the only site of synthesis of these yolk proteins (Hagedorn and Judson, 1972; Gelti-Douka et al., 1974; Chen et al., 1978). However, in D r o s o phila melanogaster, several authors reported that the synthesis of yolk polypeptides occurs both in fat body cells and in follicle cells of mature oocytes (Bownes and Hames, 1978; Jowett and Postlethwait, 1980; Brennan et al., 1982). Zhai et al. (1984) concluded that in Coccinella s e p t e m p u n c t a t a vitellogenin is produced in the fat body and in ovarian tissue. In the haemolymph of female L e p t i n o t a r s a d e c e m lineata, there are two sex specific proteins (vitellogenins) and a lipoprotein (chromoprotein 2) which are sequestered in oocytes (Peferoen et al., 1982). We now describe the onset of vitellogenesis and the time course of vitellogenin transference from the fat body to the oocytes. We also investigated whether the ovary itself is synthesizing vitellogenic and nonvitellogenic yolk proteins.
rinsed for 5 min in L. decemlineata saline and homogenized in 100/~1 0.4 M NaC1 solution. Ovarian proteins were labelled in vitro by incubating four pairs of ovaries from which all fat body tissue had been carefully removed, in 100/~1 of basal medium of Eagle with Hank's salts (Flow Laboratories) containing I0 #Ci of the previously described tritiated amino acid mixture or containing I0 pCi of [35S]methionine (1200 Ci/mmol of specific activity, Amersham). The in vitro labelling of fat body proteins was performed under the same conditions. After 5 hr the medium was collected and ovaries and fat bodies were homogenized in 200/~1 0.4 M NaCI solution. Proteins were immunoprecipitated by incubating 100 pl of the tissue culture medium with 50/~1 antiserum for 1 hr at 37°C. Goat-anti-rabbit serum (200/d) was added and this was kept at 4°C overnight. The suspension was centrifuged at 4000 rpm for 15 min. The pellet was washed twice with L. decemlineata saline and centrifuged. The final pellet was dissolved in 50/A SDS-sample buffer (Laemmli, 1970) by heating at 100°C for 15rain. Polypeptides, separated in SDS-polyacrylamide gradient (5-15%) gel electrophoresis (SDS-PAGE) with the discontinuous buffer system of Laemmli (I 970), were stained with Coomassie blue R250. Labelled polypeptides were detected by fluorography. The gel was soaked for 30 min in a 1 M sodium salicylate solution, dried on a slab gel dryer (Chamberlain, 1979) and pressed against Kodak X-Omat film for exposure at -70°C.
MATERIALS AND METHODS
RESULTS
Reproducing females were obtained by rearing ab ovo under long day conditions (LD: 25°C and 16hr photophase). Fresh potato foliage was provided daily. Female beetles were surface sterilized by rinsing for 1 min in a 1% HgCI2 solution and by dipping in two successive baths of ethanol (70 and 95% respectively). The beetles were then rinsed in distilled water, which had been sterilized by filtration (0.22 #m filter, Millipore), and were then dissected in Leptinotarsa decemlineata saline (Khan et al., 1982). Proteins were labelled in vivo by injecting 4 #Ci (4/~1) of a tritiated amino acid mixture (leucin~lysine-phenylalanine-proline-tyrosine, l mCi/ml, 70-190Ci/mmol of specific activity, Amersham). After varying time intervals (15-120 min), 1 #1 of haemolymph, about 2 mg of fat body and one ovarium, were collected. Ovaries and fat body were
The protein patterns of the ovaries, homogenized during the first days of the reproductive life, indicate the time course of yolk protein accumulation by the ovaries (Fig. 1). Shortly after eclosion (0 LD) chromoprotein 2 is one of the major proteins in the haemolymph. Until the first egg is laid (day 6), the concentration of chromoprotein 2 in the haemolymph is gradually increasing. Yet, the amount of chromoprotein 2 in the ovary is very low until the fourth day. Hereafter, there is a strong accumulation of this chromoprotein 2. The first two days after eclosion, no vitellogenins can be detected in the 251
252
M. PEFEROENand A. r)E LOOF
IR2 P.s
~b .oiov~Fb OLD
.e ov F b t . o 2LD
OVlF~ "o OV
4LD
6LD
Fig. 1. SDS-PAGE of fat body (Fb), haemolymph (He) and ovarian (OV) polypeptides of 0, 2, 4 and 6 LD females. CHR 2: chromoprotein 2, V.P.'s: vitellin polypeptides.
in
Fb He 15 mln
Ov
vivo
Fb He 30 min
protein
Ov
labelling
Fb He 60 min
Ov
Fb
He Ov 120 mt~
Fig. 2. Fluorographies of in vivo labelled fat body (Fb), haemolymph (He) and ovarian (Ov) polypeptides, separated by SDS-PAGE and visualized by fluorography. Samples were collected at different time intervals, varying from 15 to 120 min after the injection of tritiated amino acid. ~ : yolk polypeptides.
Synthesis of yolk proteins in Leptinotarsa decemlineata Vb in vitro
253
Ov in vitro
|
Fb
He
in vivo
I
CHR2
VP1
VP2
Ov in vivo
CHR2 I VP2 VP1
Fig. 3. Fluorography of in vivo labelled fat body (Fb), haemolymph (He) and ovarian (Ov) polypeptides, separated by SDS-PAGE. Immunoprecipitation of in vitro labelled fat body (Fb) and ovarian (Ov) proteins with anti-CHR 2, anti-VP 1 and anti-VP 2 sera, separated by SDS-PAGE and visualized by ftuorography. --.: yolk polypeptides.
female haemolymph but from day 3 on, vitellogenins Additional information on the origin of the yolk become highly concentrated. Yet, at that time, there proteins comes from in vitro incubations of fat body are merely traces of vitellins in the ovarian homoge- and ovaries. Isolated fat body of egg laying females nate. The accumulation of the vitellogenins by the was incubated in a tissue culture medium with radioovary starts at day 4 and continues for several weeks. labelled amino acids. Newly synthesized yolk polyApart from the onset of vitellogenesis, we also peptides were identified in the medium by immuwanted to ascertain at what rate vitellogenins are noprecipitation, SDS-PAGE and fluorography (Fig. transported from the fat body through the hae- 3). The molecular weight of in vitro synthesized yolk molymph to the ovary. For that purpose, we injected polypeptides does not differ from in vivo synthesized tritiated amino acids into the abdomen of 7 LD and circulating yolk polypeptides. females. Fat body, haemolymph and ovary samples After careful removal of all fat body tissue, ovariwere taken after different time intervals, varying oles of egg laying females were also radiolabelled in between 15 min and 2 hr. The labelled polypeptides vitro. Yolk polypeptides released into the medium were separated by SDS-PAGE and visualized by were isolated by immunoprecipitation and visualized fluorography (Fig. 2). Fifteen minutes after the injec- by SDS-PAGE and fluorography (Fig. 3). Apart tion, the radioactivity is concentrated in the vitel- from some aspecific precipitation, present in the three logenic polypeptides of the fat body. In the hae- samples, we can clearly identify labelled chromomolymph, one vitellogenic polypeptide is also weakly protein 2, vitellin 1 and vitellin 2 subunits. Chromolabelled but no labelling can be found in the vitellins. protein 2 is synthesized by the ovary from day 0 on, Except for the chromoprotein 2 and the vitellin while in vitro labelled vitellogenic polypeptides can be polypeptides, many polypeptides of the ovary are identified from day 4 on. The yolk polypeptides labelled after 15 min incorporation time. After 30 min synthesized by the ovary could not be discriminated of incorporation, the vitellogenic polypeptides in the from the yolk polypeptides synthesized by the fat haemolymph are intensely labelled but there are still body. no labelled vitellins. One hour after the injection, the amount of labelled vitellogenins in the fat body is DISCUSSION strongly reduced, while labelled vitellins appear in the ovary. One hour later, the amount of labelled vitelAlthough both vitellogenins and the main haelogenins in the haemolymph has reached its maximal molymph lipoprotein (chromoprotein 2) are highly value and newly synthesized vitellins are intensely concentrated in the haemolymph of 4 LD females, accumulated by the ovary. At the same time, the only low amounts of these proteins can be detected chromoprotein 2 in the ovary is also clearly labelled. in the ovary. A time lag between vitellogenin syn-
254
M. PEFEROENand A. DE Looo
thesis and yolk sequestration might indicate that both ing adult life, the mass of fat body tissue is gradually processes are independently controlled. This was also reduced (unpublished observation) so that the prodsuggested in Drosophila melanogaster (Postlethwait uction of yolk proteins by the ovary may become of and Shirk, 1981) and in Oncopeltus faseiatus (Kelly more importance. In Coccinella septempunctata, the and Hunt, 1982). Apparently, in young oocytes (less same hypothesis was postulated by Zhai et al. (1984), than 4 days) the follicle epithelium and/or the oocyte Brennan et al. (1982) demonstrated that in Drosomembrane are not yet capable of accumulating pro- phila melanogaster vitellogenin synthesis occurs in teins. ovarian follicle cells. The follicle cells of Leptinotarsa The fat body of 7 LD females synthesizes high decemlineata are the only cells in the ovary where amounts of vitellogenin polypeptides. Newly syn- extensive rough endoplasmatic reticulum and Golgi thesized vitellogenin polypeptides are almost immedi- complexes occur (De Loof and Lagasse, 1970). This ately released into the haemolymph, since 15 rain suggests, but does not prove, that they are the site of after the injection of tritiated amino acids, labelled endogenous yolk protein synthesis. vitellogenin polypeptides are present in the haemolymph. At that time, many ovarian polypeptides REFERENCES are labelled and since they do not correspond to labelled polypeptides in the haemolymph, we assume Bownes M. and Hames B. D. (1978) Analysis of the yolk protein in Drosophila melanogaster. Translation in a cell that they are synthesized by the ovary itself. One hour free system and peptide analysis. FEBS Lett. 96, 327-330. after the injection, most of the labelling is found in Brennan M. D., Weiner A. J., Goralski T. J. and Mahowald the vitellogenin polypeptides circulating in the haeA. P. (1982) The follicle cells are a major site of vitelmolymph. Labelled vitellin polypeptides now appear logenin synthesis in Drosophila melanogaster. Devl Biol. in the ovary. One hour later, the amount of labelled 89, 225-236. vitellogenin polypeptides has reached its maximum in Chamberlain J. P. (1979) Fluorographic detection of radiothe haemolymph while labelled vitellin polypeptides activity in polyacrylamide gels with the water-soluble are clearly accumulated by the oocyte. The incorpofluor, sodium salicylate. Analyt. Biochem. 98, 132-135. ration of labelled amino acids into proteins by the fat Chen T. T., Strahlendorf P. W. and Wyatt G. R. (1978) Vitellin and vitellogenin from locusts (Locusta mibody has almost come to an end, which is probably gratoria). Properties and post-translational modification due to the depletion of all tritiated amino acids. in the fat body. J. biol. Chem. 253, 5325 5331. Chromoprotein 2 is synthesized at a significantly lower rate than the vitellogenins. The mol. wts of De Loof A. and Lagasse A. (1970) The ultrastructure of the folliclecells of the ovary of the Colorado beetle in relation the vitellogenic polypeptides in the fat body and to yolk formation. J. Insect Physiol. 16, 211-220. haemolymph are identical. The molecular weight Gelti-Douka H., Gingeras T. R. and Kambysellis M. P. differences between vitellogenin polypeptides and (1974) Yolk proteins in Drosophila: identification and site vitellin polypeptides (Fig. 2) are caused by overof synthesis. J. exp. Zool. 187, 167-172. loading the gel and do not represent actual molecular Hagedorn H. H. and Judson C. L. (1972) Purification and site of synthesis of Aedes aegypti yolk proteins. J. exp. weight differences. There is however, a difference in Zool. 182, 367-378. molecular weight, but not in immunoreactivity, beJowett T. and Postlethwait J. H. (1980) The regulation of tween the chromoprotein 2 in the fat body and the yolk polypeptide synthesis in Drosophila ovaries and fat haemolymph, indicating processing of this protein. body by 20-hydroxy-ecdysone and a juvenile hormone This however, needs further research and is beyond analog. Devl Biol. 80, 225-234. the scope of this article. Kelly T. J. and Hunt L. M. (1982) Endocrine influenceupon The in vivo protein labelling clearly demonstrates a the development of vitellogeniccompetency in Oncopeltus faseiatus. J. Insect Physiol. 28, 935-941. very high synthesis of vitellogenins by the fat body and the uptake of these proteins by the ovary, within Khan M. A., Doderer A., Koopmanschap A. B. and de Kort C. A. D. (1982) Improved assay conditions for 1 hr after their production. We cannot exclude the in measurements of corpus allatum activity in vitro in the vivo synthesis of vitellogenins nor chromoprotein 2 by adult Colorado potato beetle, Leptinotarsa decemlineata. the ovary itself, but if there is, its contribution is very J. Insect Physiol. 28, 279-284. low when compared to the production by the fat Laemmli U. K. (1970) Cleavage of structural proteins body. during assembly of the head of bacteriophage T4. Nature, The in vitro experiments indicate that yolk polyLond. 227, 680~95. peptides are synthesized as well by isolated fat body Peferoen M., Stynen D. and De Loof A. (1982) A reas by the ovaries of vitellogenic females. SDS-PAGE examination of the protein pattern of the haemolymph of Leptinotarsa decemlineata, with special reference to viteldid not reveal any difference between yolk polylogenins and diapause proteins. Comp. Biochem. Physiol. peptides synthesized and secreted by fat body or by 72B, 345-351. ovaries. The results of our in vivo and in vitro experiments Postlethwait J. H. and Shirk P. D. (1981) Genetic and endocrine regulation of vitellogenesisin Drosophila. Am. clearly indicate that in Leptinotarsa decemlineata Zool. 21, 687-700. most of the yolk proteins are synthesized by the fat Zhai Q. H., Postlethwait J. H. and Bodley J. W. (1984) body. There may, however, be a small contribution of Vitellogenin synthesis in the lady beetle Coccinella sepovarian tissue to total yolk protein production. Durtempunctata. Insect Biochem. 14, 299 305.
0305-0491/86 $3.00+0.00 © 1986 Pergamon Press Ltd
SYNTHESIS OF VITELLOGENIC A N D N O N - V I T E L L O G E N I C YOLK PROTEINS BY THE F A T B O D Y A N D THE O V A R Y OF LEPTINOTARSA DECEMLINEATA M. PEFEROEN and A. DE LOOF Catholic University of Leuven, Zoological Institute, Naamsestraat 59, 3000 Leuven, Belgium (Tel: 016-22-2319) (Received 28 June 1985)
Abstract--1. Isolated ovaries of egg laying females synthesize and secrete three yolk proteins (two vitellogenins and chromoprotein 2). 2. The contribution of ovarian tissue to total yolk protein production is very small, the major site of synthesis of the three yolk proteins being the fat body. 3. There is a time lag between yolk protein synthesis by the fat body and yolk protein sequestration by the ovary. 4. In egg laying females, within 1 hr after the synthesis of both vitellogenins by the fat body, they appear in the oocytes as vitellins.
INTRODUCTION Sexually mature female insects synthesize some particular proteins which circulate in the haemolymph and are selectively accumulated by the oocytes. Until the late seventies, it was generally assumed that the fat body was the only site of synthesis of these yolk proteins (Hagedorn and Judson, 1972; Gelti-Douka et al., 1974; Chen et al., 1978). However, in D r o s o phila melanogaster, several authors reported that the synthesis of yolk polypeptides occurs both in fat body cells and in follicle cells of mature oocytes (Bownes and Hames, 1978; Jowett and Postlethwait, 1980; Brennan et al., 1982). Zhai et al. (1984) concluded that in Coccinella s e p t e m p u n c t a t a vitellogenin is produced in the fat body and in ovarian tissue. In the haemolymph of female L e p t i n o t a r s a d e c e m lineata, there are two sex specific proteins (vitellogenins) and a lipoprotein (chromoprotein 2) which are sequestered in oocytes (Peferoen et al., 1982). We now describe the onset of vitellogenesis and the time course of vitellogenin transference from the fat body to the oocytes. We also investigated whether the ovary itself is synthesizing vitellogenic and nonvitellogenic yolk proteins.
rinsed for 5 min in L. decemlineata saline and homogenized in 100/~1 0.4 M NaC1 solution. Ovarian proteins were labelled in vitro by incubating four pairs of ovaries from which all fat body tissue had been carefully removed, in 100/~1 of basal medium of Eagle with Hank's salts (Flow Laboratories) containing I0 #Ci of the previously described tritiated amino acid mixture or containing I0 pCi of [35S]methionine (1200 Ci/mmol of specific activity, Amersham). The in vitro labelling of fat body proteins was performed under the same conditions. After 5 hr the medium was collected and ovaries and fat bodies were homogenized in 200/~1 0.4 M NaCI solution. Proteins were immunoprecipitated by incubating 100 pl of the tissue culture medium with 50/~1 antiserum for 1 hr at 37°C. Goat-anti-rabbit serum (200/d) was added and this was kept at 4°C overnight. The suspension was centrifuged at 4000 rpm for 15 min. The pellet was washed twice with L. decemlineata saline and centrifuged. The final pellet was dissolved in 50/A SDS-sample buffer (Laemmli, 1970) by heating at 100°C for 15rain. Polypeptides, separated in SDS-polyacrylamide gradient (5-15%) gel electrophoresis (SDS-PAGE) with the discontinuous buffer system of Laemmli (I 970), were stained with Coomassie blue R250. Labelled polypeptides were detected by fluorography. The gel was soaked for 30 min in a 1 M sodium salicylate solution, dried on a slab gel dryer (Chamberlain, 1979) and pressed against Kodak X-Omat film for exposure at -70°C.
MATERIALS AND METHODS
RESULTS
Reproducing females were obtained by rearing ab ovo under long day conditions (LD: 25°C and 16hr photophase). Fresh potato foliage was provided daily. Female beetles were surface sterilized by rinsing for 1 min in a 1% HgCI2 solution and by dipping in two successive baths of ethanol (70 and 95% respectively). The beetles were then rinsed in distilled water, which had been sterilized by filtration (0.22 #m filter, Millipore), and were then dissected in Leptinotarsa decemlineata saline (Khan et al., 1982). Proteins were labelled in vivo by injecting 4 #Ci (4/~1) of a tritiated amino acid mixture (leucin~lysine-phenylalanine-proline-tyrosine, l mCi/ml, 70-190Ci/mmol of specific activity, Amersham). After varying time intervals (15-120 min), 1 #1 of haemolymph, about 2 mg of fat body and one ovarium, were collected. Ovaries and fat body were
The protein patterns of the ovaries, homogenized during the first days of the reproductive life, indicate the time course of yolk protein accumulation by the ovaries (Fig. 1). Shortly after eclosion (0 LD) chromoprotein 2 is one of the major proteins in the haemolymph. Until the first egg is laid (day 6), the concentration of chromoprotein 2 in the haemolymph is gradually increasing. Yet, the amount of chromoprotein 2 in the ovary is very low until the fourth day. Hereafter, there is a strong accumulation of this chromoprotein 2. The first two days after eclosion, no vitellogenins can be detected in the 251
252
M. PEFEROENand A. r)E LOOF
IR2 P.s
~b .oiov~Fb OLD
.e ov F b t . o 2LD
OVlF~ "o OV
4LD
6LD
Fig. 1. SDS-PAGE of fat body (Fb), haemolymph (He) and ovarian (OV) polypeptides of 0, 2, 4 and 6 LD females. CHR 2: chromoprotein 2, V.P.'s: vitellin polypeptides.
in
Fb He 15 mln
Ov
vivo
Fb He 30 min
protein
Ov
labelling
Fb He 60 min
Ov
Fb
He Ov 120 mt~
Fig. 2. Fluorographies of in vivo labelled fat body (Fb), haemolymph (He) and ovarian (Ov) polypeptides, separated by SDS-PAGE and visualized by fluorography. Samples were collected at different time intervals, varying from 15 to 120 min after the injection of tritiated amino acid. ~ : yolk polypeptides.
Synthesis of yolk proteins in Leptinotarsa decemlineata Vb in vitro
253
Ov in vitro
|
Fb
He
in vivo
I
CHR2
VP1
VP2
Ov in vivo
CHR2 I VP2 VP1
Fig. 3. Fluorography of in vivo labelled fat body (Fb), haemolymph (He) and ovarian (Ov) polypeptides, separated by SDS-PAGE. Immunoprecipitation of in vitro labelled fat body (Fb) and ovarian (Ov) proteins with anti-CHR 2, anti-VP 1 and anti-VP 2 sera, separated by SDS-PAGE and visualized by ftuorography. --.: yolk polypeptides.
female haemolymph but from day 3 on, vitellogenins Additional information on the origin of the yolk become highly concentrated. Yet, at that time, there proteins comes from in vitro incubations of fat body are merely traces of vitellins in the ovarian homoge- and ovaries. Isolated fat body of egg laying females nate. The accumulation of the vitellogenins by the was incubated in a tissue culture medium with radioovary starts at day 4 and continues for several weeks. labelled amino acids. Newly synthesized yolk polyApart from the onset of vitellogenesis, we also peptides were identified in the medium by immuwanted to ascertain at what rate vitellogenins are noprecipitation, SDS-PAGE and fluorography (Fig. transported from the fat body through the hae- 3). The molecular weight of in vitro synthesized yolk molymph to the ovary. For that purpose, we injected polypeptides does not differ from in vivo synthesized tritiated amino acids into the abdomen of 7 LD and circulating yolk polypeptides. females. Fat body, haemolymph and ovary samples After careful removal of all fat body tissue, ovariwere taken after different time intervals, varying oles of egg laying females were also radiolabelled in between 15 min and 2 hr. The labelled polypeptides vitro. Yolk polypeptides released into the medium were separated by SDS-PAGE and visualized by were isolated by immunoprecipitation and visualized fluorography (Fig. 2). Fifteen minutes after the injec- by SDS-PAGE and fluorography (Fig. 3). Apart tion, the radioactivity is concentrated in the vitel- from some aspecific precipitation, present in the three logenic polypeptides of the fat body. In the hae- samples, we can clearly identify labelled chromomolymph, one vitellogenic polypeptide is also weakly protein 2, vitellin 1 and vitellin 2 subunits. Chromolabelled but no labelling can be found in the vitellins. protein 2 is synthesized by the ovary from day 0 on, Except for the chromoprotein 2 and the vitellin while in vitro labelled vitellogenic polypeptides can be polypeptides, many polypeptides of the ovary are identified from day 4 on. The yolk polypeptides labelled after 15 min incorporation time. After 30 min synthesized by the ovary could not be discriminated of incorporation, the vitellogenic polypeptides in the from the yolk polypeptides synthesized by the fat haemolymph are intensely labelled but there are still body. no labelled vitellins. One hour after the injection, the amount of labelled vitellogenins in the fat body is DISCUSSION strongly reduced, while labelled vitellins appear in the ovary. One hour later, the amount of labelled vitelAlthough both vitellogenins and the main haelogenins in the haemolymph has reached its maximal molymph lipoprotein (chromoprotein 2) are highly value and newly synthesized vitellins are intensely concentrated in the haemolymph of 4 LD females, accumulated by the ovary. At the same time, the only low amounts of these proteins can be detected chromoprotein 2 in the ovary is also clearly labelled. in the ovary. A time lag between vitellogenin syn-
254
M. PEFEROENand A. DE Looo
thesis and yolk sequestration might indicate that both ing adult life, the mass of fat body tissue is gradually processes are independently controlled. This was also reduced (unpublished observation) so that the prodsuggested in Drosophila melanogaster (Postlethwait uction of yolk proteins by the ovary may become of and Shirk, 1981) and in Oncopeltus faseiatus (Kelly more importance. In Coccinella septempunctata, the and Hunt, 1982). Apparently, in young oocytes (less same hypothesis was postulated by Zhai et al. (1984), than 4 days) the follicle epithelium and/or the oocyte Brennan et al. (1982) demonstrated that in Drosomembrane are not yet capable of accumulating pro- phila melanogaster vitellogenin synthesis occurs in teins. ovarian follicle cells. The follicle cells of Leptinotarsa The fat body of 7 LD females synthesizes high decemlineata are the only cells in the ovary where amounts of vitellogenin polypeptides. Newly syn- extensive rough endoplasmatic reticulum and Golgi thesized vitellogenin polypeptides are almost immedi- complexes occur (De Loof and Lagasse, 1970). This ately released into the haemolymph, since 15 rain suggests, but does not prove, that they are the site of after the injection of tritiated amino acids, labelled endogenous yolk protein synthesis. vitellogenin polypeptides are present in the haemolymph. At that time, many ovarian polypeptides REFERENCES are labelled and since they do not correspond to labelled polypeptides in the haemolymph, we assume Bownes M. and Hames B. D. (1978) Analysis of the yolk protein in Drosophila melanogaster. Translation in a cell that they are synthesized by the ovary itself. One hour free system and peptide analysis. FEBS Lett. 96, 327-330. after the injection, most of the labelling is found in Brennan M. D., Weiner A. J., Goralski T. J. and Mahowald the vitellogenin polypeptides circulating in the haeA. P. (1982) The follicle cells are a major site of vitelmolymph. Labelled vitellin polypeptides now appear logenin synthesis in Drosophila melanogaster. Devl Biol. in the ovary. One hour later, the amount of labelled 89, 225-236. vitellogenin polypeptides has reached its maximum in Chamberlain J. P. (1979) Fluorographic detection of radiothe haemolymph while labelled vitellin polypeptides activity in polyacrylamide gels with the water-soluble are clearly accumulated by the oocyte. The incorpofluor, sodium salicylate. Analyt. Biochem. 98, 132-135. ration of labelled amino acids into proteins by the fat Chen T. T., Strahlendorf P. W. and Wyatt G. R. (1978) Vitellin and vitellogenin from locusts (Locusta mibody has almost come to an end, which is probably gratoria). Properties and post-translational modification due to the depletion of all tritiated amino acids. in the fat body. J. biol. Chem. 253, 5325 5331. Chromoprotein 2 is synthesized at a significantly lower rate than the vitellogenins. The mol. wts of De Loof A. and Lagasse A. (1970) The ultrastructure of the folliclecells of the ovary of the Colorado beetle in relation the vitellogenic polypeptides in the fat body and to yolk formation. J. Insect Physiol. 16, 211-220. haemolymph are identical. The molecular weight Gelti-Douka H., Gingeras T. R. and Kambysellis M. P. differences between vitellogenin polypeptides and (1974) Yolk proteins in Drosophila: identification and site vitellin polypeptides (Fig. 2) are caused by overof synthesis. J. exp. Zool. 187, 167-172. loading the gel and do not represent actual molecular Hagedorn H. H. and Judson C. L. (1972) Purification and site of synthesis of Aedes aegypti yolk proteins. J. exp. weight differences. There is however, a difference in Zool. 182, 367-378. molecular weight, but not in immunoreactivity, beJowett T. and Postlethwait J. H. (1980) The regulation of tween the chromoprotein 2 in the fat body and the yolk polypeptide synthesis in Drosophila ovaries and fat haemolymph, indicating processing of this protein. body by 20-hydroxy-ecdysone and a juvenile hormone This however, needs further research and is beyond analog. Devl Biol. 80, 225-234. the scope of this article. Kelly T. J. and Hunt L. M. (1982) Endocrine influenceupon The in vivo protein labelling clearly demonstrates a the development of vitellogeniccompetency in Oncopeltus faseiatus. J. Insect Physiol. 28, 935-941. very high synthesis of vitellogenins by the fat body and the uptake of these proteins by the ovary, within Khan M. A., Doderer A., Koopmanschap A. B. and de Kort C. A. D. (1982) Improved assay conditions for 1 hr after their production. We cannot exclude the in measurements of corpus allatum activity in vitro in the vivo synthesis of vitellogenins nor chromoprotein 2 by adult Colorado potato beetle, Leptinotarsa decemlineata. the ovary itself, but if there is, its contribution is very J. Insect Physiol. 28, 279-284. low when compared to the production by the fat Laemmli U. K. (1970) Cleavage of structural proteins body. during assembly of the head of bacteriophage T4. Nature, The in vitro experiments indicate that yolk polyLond. 227, 680~95. peptides are synthesized as well by isolated fat body Peferoen M., Stynen D. and De Loof A. (1982) A reas by the ovaries of vitellogenic females. SDS-PAGE examination of the protein pattern of the haemolymph of Leptinotarsa decemlineata, with special reference to viteldid not reveal any difference between yolk polylogenins and diapause proteins. Comp. Biochem. Physiol. peptides synthesized and secreted by fat body or by 72B, 345-351. ovaries. The results of our in vivo and in vitro experiments Postlethwait J. H. and Shirk P. D. (1981) Genetic and endocrine regulation of vitellogenesisin Drosophila. Am. clearly indicate that in Leptinotarsa decemlineata Zool. 21, 687-700. most of the yolk proteins are synthesized by the fat Zhai Q. H., Postlethwait J. H. and Bodley J. W. (1984) body. There may, however, be a small contribution of Vitellogenin synthesis in the lady beetle Coccinella sepovarian tissue to total yolk protein production. Durtempunctata. Insect Biochem. 14, 299 305.