vitellogenesis in the stick insect carausius morosus i. specific protein ...

J. Cell Sri. 46, 1-16 (1980) Printed in Great Britain © Company of Biologists Limited 1980

VITELLOGENESIS IN THE STICK INSECT CARAUSIUS MOROSUS I. SPECIFIC PROTEIN SYNTHESIS DURING OVARIAN DEVELOPMENT FRANCO GIORGI AND FAUSTO MACCHI Istituto di Istologia e Embriologia, Universitd di Pisa

SUMMARY Vitellogenesis in the stick insect Carausius morosus (Br.) has been studied with the goal of identifying vitellogenin in various tissues. Following exposure in vivo to radioactive amino acids, oocytes in the medium size range are labelled with a minimum delay of 6 h after the time of injection. Incorporation of radioactivity under these conditions is shown to depend upon accumulation of proteins rather than on a differential rate of protein synthesis in succeeding stages -of oogenesis. By immunochemical analyses, it is shown that at least two antigens are common to both haemolymph and ovary and that one of these is also present in the fat body. Both antigens are labelled during exposure to radioactive amino acids. When analysed by SDS polyacrylamide gel electrophoresis, extracts from both haemolymph and ovary appear to share a number of protein fractions which range in molecular weight from 40000 to 200000 Daltons. The labelling pattern exhibited by these fractions is clearly indicativ e of a protein transfer from the fat body to the oocyte. Fat body cultured in vitro for up to 4 h releases a major macromolecular complex in the external medium. The latter has been identified as vitellogenin by both immunoprecipitation assay and SDS polyacrylamide gel electrophoresis. The protein which is synthesized and secreted under these conditions results from the processing of a protein complex of higher molecular weight.

INTRODUCTION

Vitellogenesis is a key event in ovarian development. It occurs by synthesis of a specific yolk precursor, vitellogenin, in a tissue other than the ovary, namely, the fat body (Brookes, 1969; Pan, 1971). Vitellogenin is not stored to any extent but is rather secreted into the maternal haemolymph soon after being synthesized by the producing tissue (Pan, Bell & Telfer, 1969). Turnover of vitellogenin in the haemolymph is thought to result from a balanced equilibrium between the rate of secretion by the fat body and that of the intake process by the oocyte (Buhlmann, 1976; Kambysellis, 1977). By virtue of both these processes, vitellogenic oocytes can engulf large amounts of vitellogenin and store it in a molecular form made insoluble by a mechanism as yet not fully understood (Bergink & Wallace, 1974; Giorgi & Jacob, 1977). Apparently, it looks as though vitellogenin may escape degradation only when taken into the oocyte by pinocytosis (Giorgi, 1980) and stored in the socalled yolk spheres (Wallace & Hollinger, 1979). Injected vitellogenin is, in fact, degraded by the oocyte in a manner not dissimilar from that of other serum proteins (Dehn & Wallace, 1973). ConAuthor's address: Dr Franco Giorgi, Istituto di Istologia e Embriologia, Via A. Volta 4, 56100-Pisa, Italy.

2

F. Giorgi and F. Macchi

sequently, yolk proteins may be stored unmodified by the oocyte, and so made available to the developing embryo after fertilization, only if maintained in a membrane-bound cell compartment (Lemansky & Aldoroty, 1977; Lundquist & Emanuelsson, 1979). In order for an oocyte to accomplish vitellogenesis, several basic conditions have to be fulfilled. First, vitellogenin titre in the haemolymph has to be sufficiently high to sustain vitellogenic maturation of all developing oocytes. Secondly, the oocyte itself has to become competent to single out vitellogenin from the circulating fluid (Giorgi, 1979). Finally, these 2 functions have to be kept in equilibrium with each other so as to keep ovarian development in balance with the animal's general nourishment (Hagedorn & Fallon, 1973; Bosquet, 1979) and with photoperiodicity (Dortland, 1978). In most animal species, any one of these functions is under hormonal control (Postlethwait & Weiser, 1973; Sakurai, 1977). In fact, we know that synthesis and uptake of vitellogenin are both triggered by availability of juvenile hormone in the haemolymph and that ecdysone may also play a role in these processes (Pan & Wyatt, 1971; Engelman, 1969; Handler & Postlethwait, 1978; Bell & Barth, 1971; Hagedorn, Fallon & Laufer, 1973). Although the scheme so far described may be applicable to most of the oviparous species with good approximation, including non-mammalian vertebrates (Wallace, 1978), there seems to be an increasing demand for extending our present knowledge to species other than those which are currently used as models for developmental biology. Based on these considerations we thought it of interest to extend our investigation on vitellogenesis to a parthenogenetic species, as is the case for Carausius morosus, for which such aspects as sex determination and genetic control of vitellogenesis may be expected to be different from those of amphigonic species (Koch, 1964). In this first paper we report an investigation of the pattern of protein synthesis by several tissues. On the basis of the criteria outlined above, this study aims to identify vitellogenin, its precursor(s), if any, and its processed products in these tissues. It is thus intended to serve as a possible basis for purification and further biochemical analysis of the vitellogenin molecule. MATERIAL AND METHODS Specimens of the parthenogenetic species Carausius morosus (Br.) were reared in perspex cages and maintained on a diet of fresh ivy leaves. Females of similar age and showing a comparable egg producing rate were used throughout this experimental work. Prior to dissection, animals were anaesthetized in a gaseous flow of CO|. Haemolymph was drawn from the animal by using a sterile plastic syringe. To prevent haemolymph clotting, which is known to occur upon exposure to air (Brehelin, 1979), haemolymph was diluted with 1 vol. of 1 mM EDTA in 10 % glucose solution. Ovarioles were removed from the abdominal cavity while the animal was maintained in physiological saline solution. They were then transferred to a small plastic Petri dish, divested of their enveloping layers and measured under a stereo-microscope using a calibrated eye piece. The oocyte volume at each developmental stage was calculated on the assumption that the oocyte corresponds to a rotational ellipsoid. Accordingly, only the 2 major axes of each oocyte were measured. Following complete removal of all ovariole and intestine, fat body was collected by scraping the internal side of the abdominal body wall with

Vitellogenesis in Carausius

3

a small spatula. Extracts from both ovarioles and fat body were prepared by homogenization in 0-05 M Tris-HCl buffer at pH 6-8. Tissue extracts were stored in small aliquots at — 20 °C until used for analysis. Protein concentration was determined according to Lowry, Rosebrough, Farr & Randall (1951) using bovin serum albumin as a standard. Immunochemical analyses Antibodies to haemolymph proteins (E) and to egg proteins (O) were raised in rabbits by injecting, subcutaneously and intramuscularly, 30 mg of antigen emulsified with an equal amount of complete Freund's adjuvant. Following a second injection of antigen emulsified with incomplete Freund's adjuvant, the rabbits then underwent a weekly series of alternative bleedings and intravenous antigen injections. After blood clotting, rabbit antiserum was used for immunological analyses with no further purification. Tissue extracts were analysed against their respective antisera by double immunodiffusion (Ouchterlony, 1968), by immunoelectrophoresis on agar plates (Grabar & Williams, 1953) and by rocket immunoelectrophoresis (Laurell, 1966). Incorporation of radioactivity For incorporation studies, animals were injected with 5 fid of fH] aspartic acid (sp. act. 178 Ci/mM) and/or 70/iCi of [^SJmethionine (sp. act. 1020 Ci/mM) (Radiochemical Centre. Amersham). After varying times of exposure to the radioisotopes - 1 , 3 , 6 , 12, 18, 24 or 48 h tissues were dissected out and treated as described below. Incorporation of radioactivity into oocytes was assessed by placing each oocyte, previously measured with a stereomicroscope, in a separate scintillation vial; this was followed by overnight digestion in 05 ml of Soluene (Packard) and counting in a Beckman LS-100C counter. Radioactivity of TCA-precipitable material was determined by placing aliquots of tissue extracts on paper disks (3 MM Whatman); these were treated with hot 100 % trichloroacetic acid (TCA) for 5 min, washed twice with ethanol-ether, washed again in ether and finally dried (Mans & Novelli, 1961). The paper disks were placed in scintillation vials with 8 ml of toluene containing 0-32 % diphenyloxazole (PPO), 0-02% i,4&is-[2(4-methyl-5-phenyloxazolyl)]-benzene (POPOP) and counted in a Beckman LS-100C counter. In vitro culturing Fat body was excised from the abdominal body wall as already described and cultured in Grace's medium (Gibco) for periods of 1, 2 or 4 h. Ten microCuries of fHJaspartic acid were added to 1 ml of culture medium supplemented with 50 fil of fresh haemolymph. All glassware used in this experiment was made sterile by overnight exposure to an ultraviolet lamp and incubation media were sterile filtered. For each time point, aliquots of 100 fii of culture medium were incubated overnight with 100 pX of anti-egg antiserum. To check for specificity of the reaction, the immunoprecipitate was first washed in 005 M Tris-HCl buffer at pH 6'8 and then dissolved in 2 % sodium, dodecyl sulphate (SDS) solution in the same buffer. Finally, it was analysed by 10% SDS-polyacrylamide gel electrophoresis and processed for fluorography as described below. Electrophoresis and fluorography 10 % SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was carried out in a vertical slab gel apparatus according to the procedure of Laemmli (1970). Tissue extracts were diluted with an equal volume of SDS sample buffer (2 % SDS in 0-05 M Tris-HCl buffer at pH 6-8 containing 5 % mercapto-ethanol), boiled for 3 min and then stored in small aliquots until used. Molecular weights of major protein fractions from haemolymph and oocyte were determined by comparison with standard proteins of known molecular weight which were run in the same gel. Sample and standard proteins were electrophoresed, under the same electrical conditions, on gel tubes of varying polyacrylamide concentrations ( T = 5, 10, or 15 %) and in each case a plot of molecular weights versus their relative electrophoretic mobility was made. Upon termination of electrophoresis, polyacrylamide gels were stained overnight in 1 % Coomassie brilliant blue G250 in 45 % ethanol-10 % acetic acid and destained through several changes in

4


the same solution. Since sample proteins were electrophoresed in duplicate for each exposure time, the stained gels could be cut in half and each part treated differently. For autoradiography, half of the gel was dried under vacuum, mounted with Agfa-Gaevert X-ray films and exposed in light-proof boxes for periods of time ranging from one to several weeks. Fluorography was carried out by dehydrating half of each slab gel with dimethyJsulphoxide (DMSO) followed by impregnation in 22-2 % PPO solution (w/v) in DMSO. Once dried as above, the slab gels were exposed with an X-ray film at —70 °C for 3-14 days (Bonner & Laskey, 1974).

RESULTS

In order to assess how protein labelling occurs during ovarian development, we first examined incorporation of radioactive amino acids into oocytes of varying sizes. Fig. 1 shows that, up to 6 h of in vivo exposure to [^HJaspartic acid, oocytes at all developmental stages appear to incorporate radioactivity at a more or less similar rate. From 12 h onwards, however, oocytes of medium sizes - ranging from 2 to 5 mm3 in volume - appear to become far more active in incorporating radioactive amino acids than those of either larger or smaller sizes. We also noted that when oocytes of these sizes are exposed in vivo for periods of time longer than 6 h, they appear to incorporate radioactivity at proportionally higher rates. In addition, as exposure in vivo is prolonged from 12 to 24 h, the volume at which oocytes exhibit the highest radioactivity incorporation becomes progressively larger. These observations imply that under our experimental conditions, incorporation into medium-size oocytes is mainly due to labelling of proteins which are not subjected to turnover. Whether this pattern of labelling is due to ovarian-synthesized proteins or to proteins taken into the oocyte from the haemolymph cannot be determined on the basis of this experiment alone. To ascertain whether a major protein fraction of the ovary does indeed accumulate, we turned to examine how medium-sized oocytes are labelled during a 48-h period of in vivo exposure to radioactive amino acids. Incorporation of [3H]aspartic acid into TCA-precipitable proteins from oocyte extracts rises linearly over the time period tested (Fig. 2 c). By comparison, pH]aspartic acid incorporation into fat body and haemolymph proteins appears to follow a steady rise for up to 24 h and then to decline rapidly afterwards (Fig. 2 A, B). While the pattern of labelling observed in ovarian tissues is clearly indicative of a steady accumulation of the radiolabelled proteins, that observed in fat body and haemolymph has to be interpreted differently. The latter could, in fact, be attributed either to exhaustion of the radioactive amino acid pool or rather to the fact that the protein(s) synthesized during the period of exposure are exported outside the tissues themselves. Although total counts in the haemolymph are still very high at the time protein labelling appears to decline, the first hypothesis cannot be completely ruled out. This could be done only by knowing the exact extent of the intracellular amino acid pool (Regier & Kafatos, 1971). To pursue further the idea of protein transport from one compartment to another of the vitellogenic system, we attempted to examine whether the selected tissues share a common protein content among themselves. As shown in Fig. 3 A, there are at least 2 major antigens which are common to both oocytes and haemolymph when extracts of these tissues are made to react with their respective antisera. When such a


5

comparison is extended to fat body extracts, it appears that at least 1 of the 2 antigens shared by oocytes and haemolymph is also present in this tissue (Fig. 3B). A similar conclusion could be drawn from the immunoelectrophoretic analysis shown in Fig. 3D, where 2 overlapped precipitation arcs may be seen. The 2 antigens shared by

1

2

3

4

5

6

7

1

2

3

4

5

6

7

Oocyte vol., mm 3

Fig. 1. Extent of amino acid incorporation as a function of oocyte volume. Oocytes were exposed in vivo to ["Hjaspartic acid and [^Slmethionine: for A, I (O), 3 ( • ) , and 6 h (A) for B, 12 ( • ) , 18 (B), and 24 h (A), and the resulting incorporation expressed as total counts over time. The ratio between incorporation of PH]aspartic acid to ["SJmethionine is also indicated ( ).

6 12 24

6 12

24

48

6 12

24

48

Exposure time, h

Fig. 2. Relative rates of PFfJaspartic acid incorporated in vivo into proteins of fat body (A), haemolymph (B), and ovary (c) as a function of time.


Fig. 3. Immunological analyses of tissue extracts from fat body (b), haemolymph (e) and ovary (ov) as tested against anti-egg (O) and anti-haemolymph (E) antisera. A, B, double immunodiffusion on i % agar plates, c, rocket immunoelectrophoresis on agarose plates containing 1/10 of anti-egg antiserum. Samples of haemolymph were exposed in vivo to [•)5S]methionine for 3,6, 12, 18, 24 or 48 h and then electrophoresed overnight in 0-05 M Tris-citric acid buffer at pH 82 at 220 V/25 mA. D, immunoelectrophoresis on agar plates. The antigens used in this analysis were exposed in vivo for 6 h to [3°S]methionine. Electrophoresis in the first dimension was performed at 200 V/ 25 mA for 30 min; subsequently, the antigens were left to diffuse overnight against their respective antisera. E, F, autoradiographs of the plates shown in C and D.


y

oocytes and haemolymph are better evidenced by analysing aliquots of these tissues by rocket immunoelectrophoresis (Fig. 3 c). Both these antigens, though variable in their respective titre, become radioactively labelled during the time of exposure to [^SJmethionine (Fig. 3E, F). Having determined that fat body, haemolymph and oocytes share part of their antigenic composition with each other and that protein labelling occurs at a rate which is sufficiently high to be studied during the periods selected for in vivo exposure, we were ready to analyse how protein fractions are labelled in each tissue. Accordingly, a number of females were injected with pH]aspartic acid and/or [^SJmethionine and, at due times, they were sacrificed and their tissues analysed. Of the 10 major protein fractions revealed by SDS polyacrylamide gel electrophoresis in oocytes, only 6 exhibit an electrophoretic mobility similar to that of the fractions present in the haemolymph (Fig. 4A). Comparison with fat body electropherogram is made difficult by the large number of protein fractions which are detectable by this technique and by the low representation of each fraction in this tissue. Since standard proteins of known molecular weight were electrophoresed along with those of the vitellogenic tissues, their shared protein fractions are, hereafter, termed by making reference to their estimated molecular weight (Fig. 4 A). The relative rates of protein synthesis in these tissues can be qualitatively estimated by comparing the set of fluorographs shown in Fig. 4B. After 1 h of exposure to PHJaspartic acid, the protein fractions which exhibit the highest specific activity in the fat body are those of 200000 and 250000 molecular weight. A protein fraction showing an apparently similar or slightly smaller molecular weight than 200000, although with less amount of label on it, is also present in the haemolymph. No protein fraction is labelled in the oocyte at this time interval. Other major protein fractions which become labelled in the haemolymph at this exposure time are the ones with molecular weights around 100 000, 125000, and 130000. The last of these protein fractions has also a counterpart in the fat body, though much less labelled than in the haemolymph. With progressively longer exposures to radioactive amino acids, the pattern of labelling in each tissue changes drastically. The essential feature of these patterns is the appearance of the 200000 protein fraction in the oocyte. This is also the only protein fraction to exhibit an increasing amount of label as time goes by. When fat body is exposed for 24 h in vivo to radioactive amino acids and then chased for up to 4 h in an in vitro system, the amount of radioactivity appearing in the medium in the form of proteins precipitable by the anti-egg antiserum increases slightly over the same time period (Fig. 5 A). A somewhat similar release of antiserumprecipitable protein could be obtained when fat body, not previously exposed to radioactive amino acids, was incubated in vitro in a radioactive medium (Fig. 5B). However, under these conditions the radioactivity incorporated into fat body proteins does not decline, but follows a pattern of incorporation more readily comparable to that detectable during the first phases of in vivo exposure (see Fig. 2 A). Analysis by SDS-polyacrylamide gel electrophoresis of the proteins immunoprecipitated from the culture medium revealed that at least 60% of the total precipitate


A

300 K.

a 4 250 K

165 K •

b « 200 K c 4 125 K • d « 100 K

155K'

e

85 K

-as.

68 K 43 K 39 K

21-5 K

B

ii

ir

I

. f 4 •g ^ h

60 K 55 K 50 K

•i

40 K


9

is represented by a protein fraction which has a molecular weight around 200000 (Fig. 6). This observation provides strong evidence in favour of the view that the protein released by the fat body in vitro is indeed the same fraction which is transferred to the maternal haemolymph and to the oocyte (Fig. 6 A). To determine whether the protein fraction released by the fat body in the culture medium results from processing of a larger polypeptide, we again cultured fragments of this tissue in a radioactive medium and then analysed their SDS-solubilized extracts by gel electrophoresis. The results of this experiment indicate that the 2 major fractions which are synthesized by the fat body under these conditions, are labelled in a sequential order (Fig. 7). In fact, as label decreases in the heavier fraction, it decreases in the lighter one within a 4-h period of in vitro culturing.

Incubation time, h

Fig. 5. Rates of radioactivity incorporation into proteins of fat body following either in vivo (A) or in vitro (B) exposure to PHJaspartic acid. Proteins released in the medium under these culturing conditions were immunoprecipitated with anti-egg antiserum and the resulting radioactivity expressed as cpm over mg of fat body protein and 100 fi\ of culture medium. Labelled proteins retained by the fat body during in vitro culturing (O—O; • — • ) and labelled proteins released in the culture medium over the same periods ( # — 9 ; • — • ) are also indicated.

Fig. 4. A, 10 % SDS-polyacrylamide gel electrophoresis of proteins from fat body (b), haemolymph (e) and ovary (ov) as compared to standard proteins of known molecular weights (M): dimer IgA, 300000 (Calbiochem.), RNA polymerase /?, 165000 (Boehring), RNA polymerase /?', 155000 (Boehring), bovine serum albumin, 68000 (Serva), ovalbumin, 43000 (Sigma), RNA polymerase a, 39000 (Boehring), trypsin inhibitor, 21500 (Boehring). Protein fractions shared by ovary and haemolymph are marked ( ^ ) . B, electrophoretic analysis (10% SDS-PAGE) of the proteins synthesized by fat body (b), haemolymph (e) and ovary (ov). Samples were electrophoresed following in vivo exposure to fHJaspartic acid for 1 (a), 3 (b) or 12 h (c), and then treated for fluorographic detection of the incorporated radioactivity. a

CEL

46


10

DISCUSSION

Vitellogenin has been traditionally defined as the sex-specific protein in the haemolymph which comprises much of the yolk proteins in the mature egg (Hagedorn & Kunkel, 1979). This definition has been questioned on the grounds that other haemolymph proteins may as well gain access to the oocyte during vitellogenesis (Engelman, 1970, 1979). In spite of this criticism, however, the term vitellogenin can still be usefully employed, provided the rate of uptake of this protein into the oocyte is carefully measured and so compared with that of other oocyte proteins having an exogenous origin (Wallace & Jared, 1976; Kunkel & Pan, 1976; Roth, Cutting & Atlas, 1976). It should be noted that such measurement becomes experimentally

Fig. 6. Densitometric scans of A, 10 % SDS-PAGE separated polypeptides from ovarian extracts, and B, fluorograph of the fat body protein released under in vitro culturing immunoprecipitated with anti-egg antisenim. Left-hand arrow, origin; right-hand arrow in A, front.

Vitellogenesis in Carausius i1 possible only when vitellogenin is obtainable in sufficiently pure form. This last consideration makes the argument on selectivity somewhat circular and so, in practice, the definition of vitellogenin is bound to rely on criteria which though useful, are not entirely satisfactory. *

A

*

B

V

c

•

Fig. 7. Densitometric scans of fluorographs of the proteins synthesized by fat body under in vitro conditions following exposure to ["HJaspartic acid for 1 (A), 2 (B), and 4 h (c). Large arrow indicates origin, small arrows indicate the 2 protein fractions which are labelled sequentially over the period tested.

This study reports on a first attempt to identify vitellogenin in the haemolymph of Carausius morosus. The criteria we have set forth in this study are based on a variety of experimental approaches all of which have to be considered as preliminary in view of the final check on selectivity. Our data show that oocytes exposed to radioactive amino acids for various lengths of time become maximally labelled in the volume range 2-5 mm3 with a minimum delay of 6 h after the time of injection. Similar observations have previously been carried out in oocytes of other insects by either autoradiographic

12


(Bier, 1962, 1963) or electrophoretic techniques (Engels, 1972). All these studies are consonant with the view that oocyte labelling is primarily due to proteins synthesized by a tissue outside the ovary and then transferred to the oocyte only after secretion into the haemolymph. The time lapse required by the oocyte to become maximally labelled can, in fact, be reasonably attributed to the time required by an extraovarian tissue to synthesize and secrete this protein and to that necessary for its transfer to the oocyte. That such a scheme could also be applied with reasonable confidence to oocyte labelling in Carausius morosus is confirmed by the observation that incorporation of radioactivity into oocytes does not turnover during the time period examined. Stability of the proteins is, in fact, a necessary though not sufficient criterion by which one can identify vitellogenin in ovarian extracts. The data on the labelling pattern of the TCA-precipitable material has in addition shown that the ovary is the only tissue of those examined to exhibit a steady rate of accumulation of radioactivity. The last consideration is also consistent with the interpretation that labelling of progressively larger oocytes, as it occurs with prolonged exposure to radioactive amino acids, is likely to depend upon accumulation of proteins rather than on a differential rate of protein synthesis in succeeding stages of vitellogenesis. To elucidate the nature of the material accumulated in oocytes, fat body and haemolymph from adult females were analysed as well. By the use of several immunological techniques we have been able to show that at least 2 antigens are common to both ovary and haemolymph and that one of these is present in the fat body. Although a common antigenic content among these tissues does not prove per se the existence of protein transfer, it is nevertheless a strong indication in favour of this interpretation. Evidence that proteins are indeed transferred from the fat body to the oocyte by way of release into the haemolymph has been obtained by the use of fluorographic and electrophoretic techniques. We have shown that each of the tissues examined by these techniques contains a predominant protein fraction with an estimated molecular weight of about 200000 which, during in vivo exposure to radioactive amino acids, is labelled in a sequential order: from fat body to oocyte. As a final experiment in our present series we have shown that the major protein fraction which is released both in vivo and in vitro by the fat body has a molecular weight which falls well within the range of that estimated for the heavily labelled fractions of the haemolymph and ovary. On the basis of the evidence so far gathered we feel confident in identifying the 200000 molecular weight protein fraction as the vitellogenin molecule, or more appropriately, as one of its major subunits. Obviously, this conclusion fails to account for 2 major aspects of the problem related to vitellogenin identification. The first of these concerns the relationship between the protein fraction resolved by gel electrophoresis under denaturing conditions and its native form. The second is whether the 2 antigenic forms, which are detected in the haemolymph by immunoelectrophoresis against anti-egg antiserum, may both be identified as native vitellogenin peptides or rather if only one of the two fits this definition. A survey of the literature on this problem shows that the vitellogenin molecule in its native form has a molecular weight of around 5 x io6 Daltons in most of the insect species so far examined (Wyatt & Pan, 1978). In addition, investigations of vitellogenin


13

substructure have revealed the presence of a varying number of subunits. For instance, 2 different subunits with respective molecular weights of 43000 and 120000 have been detected by SDS polyacrylamide gel electrophoresis in the vitellogenin molecule isolated from Cecropia pupal haemolymph (Kunkel & Pan, 1976). A similar subunit composition has been found for the vitellogenin of Philosomia cynthia, although higher molecular weights were reported for its subunits (Chino, Yamagata & Sato, 1977). A somewhat different situation has been reported in cockroaches and in the migratory locust, where 4 and 5 vitellogenin subunits have been found respectively (Clore, Petrovitch, Koeppe & Mills, 1978; Koeppe & Ofengand, 1976; Gellissen et al. 1976). Preliminary investigation in our laboratory seems to confirm the applicability of the scheme outlined above for the vitellogenin in Carausius, although as yet no certainty has been gained as to the exact number of subunits in this species. Whether 1 or 2 vitellogenin peptides exist in the native state in Carausius cannot be decided on the basis of the evidence provided by this study. Although instances of more than one vitellogenin have occasionally been reported in the literature (Hagedorn & Kunkel, 1979), we believe measurement of the relative rate of oocyte uptake for each of the haemolymph antigens is the most reliable means of solving this question. At the present time, we are inclined to believe that a unique form of vitellogenin exists in Carausius; this belief is based upon the observation that 1 haemolymph antigen is by far the most abundantly represented in ovarian extracts. In the final part of our study we demonstrated that fat body taken from adult females in Carausius is capable of releasing a labelled macromolecular complex into the external medium. Such a complex has been identified as vitellogenin by both immunoprecipitation assay and SDS-electrophoresis. The vitellogenin released under these conditions amounts to about 60% of the total protein found in the medium after 4-h culturing, implying that other haemolymph proteins may be secreted as well by the fat body during the same period of time. Similar or even higher rates of vitellogenin secretion have been previously reported for other insect species (Chen, Couble, Abu-Hakima & Wyatt, 1979). In parallel with these findings, we have also provided some evidence that the vitellogenin secreted by fat body results from a proteolytic trimming of an intracellular precursor of higher molecular weight. The evidence agrees with earlier observations in other insects (Koeppe & Ofengand, 1976; Chen, Strahlendorf & Wyatt, 1978). The processing of vitellogenin in the fat body is likely to occur prior to secretion and could thus constitute a necessary step in the release of the partially degraded product. In spite of the present incompleteness of our knowledge of the intracellular processing for secretory proteins, the evidence provided in this study, along with that by previous investigators, points to the general occurrence of such a phenomenon. Whether vitellogenin synthesis in Carausius is under hormonal control as in many other insects (Doane, 1973) or rather occurs independently of a hormonal supply as in Cecropia (Pan, 1977) could not be established beyond doubt in this study. However, our preliminary observations on isolated abdomens from adult females would seem to indicate that both synthesis and uptake of vitellogenin in Carausius occur irrespective of a hormonal control. Earlier evidence by Pflugfelder (1937) that removal of corpora

14


allata in adult Carausius has apparently no effect on vitellogenesis is also consistent with this interpretation. Perhaps it is the occurrence of continuous egg production that makes unnecessary the existence of an endocrine regulation for vitellogenin synthesis and uptake in the stick insect.

REFERENCES BELL, W. J. & BARTH, R. H. (1971). Initiation of yolk deposition by juvenile hormone. Nature, New Biol. 230, 220-221. BERGINK, E. M. & WALLACE, R. A. (1974). Precursor product relationship between amphibian vitellogenin and the yolk proteins lipovitellin and phosvitin. J. biol. Chem. 249, 2897-2903. BIER, K. (I962). AutoradiographischeUntersuchungen zur Dotterbuildung. Na*uncissenschaften 49, 332-333BIER, K. (1963). Autoradiographische Untersuchungen iiber die Leistungen des Folliklepithels und der Nahrzellen bei der Dotterbildung und Eiweissynthese im Fliegenovar. Wilhelm Roux Arch. EntwMech. Org. 154, 552-575. BONNER, W. M. & LASKEY, R. A. (1974). Afilmdetection method for tritium labelled proteins and nucleic acids in polyacrylamide gels. Eur. J. Biochem. 46, 83-88. BOSQUET, G. (1979). Occurrence of active regulatory mechanism of protein synthesis during starvation and refeeding in Bombyx mori fat body. Biochimie 61, 165-170. BREHELIN, M. (1979). Haemolymph coagulation in Locustamigratoria: evidence for a functional equivalent of fibrinogen. Comp. Biochem. Physiol. 62 B, 329-334. 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. Devi Biol. 20, 459-471. BUHLMANN, G. (1976). Haemolymph vitellogenin. Juvenile hormone and oocyte growth in the adult cockroach Nauphoeta cinerea during first pre-oviposition period. J. Insect Physiol. 22, IIOI-IIIO.

T. T., STRAHLENDORF, P. W. & WYATT, G. R. (1978). Vitellin and vitellogenin from locusts (Locusta migratoria). Properties and post-translational modification in the fat body. J. biol. Chem. 253, 5324~533i-

CHEN,

CHEN, T. T., COUBLE, P., ABU-HAKIMA, R. & WYATT, G. R. (1979). Juvenile hormone-

controlled vitellogenin synthesis in Locusta migratoria fat body. Hormonal induction in vivo. Devi Biol. 69, 59—72. CHINO, H., YAMAGATA, M. & SATO, S. (1977). Further characterization of lepidopteran vitellogenin from haemolymph and mature eggs. Insect Biochem. 7, 125-131. CLORE, J. N., PETROVITCH, E., KOEPPE, J. K. & MILLS, R. R. (1978). Vitellogenesis by the

American cockroach. Electrophoretic and antigenic characterization of haemolymph and oocyte proteins. J. Insect Physiol. 24, 45-51. DEHN, P. F. & WALLACE, R. A. (1973). Sequestered and injected vitellogenin. Alternative routes of protein processing in Xenopus laevis. J. Cell Biol. 58, 721-724. DOANE, W. W. (1973). Role of hormones in insect development. In Insects: Developmental Systems (ed. S. J. Counce & C. H. Waddington), pp. 291-497. London: Academic Press. DORTLAND, J. F. (1978). Synthesis of vitellogenins and diapause proteins by the fat body of Leptinotarsa as a function of photoperiod. Physiol. Ent. 3, 281-288. ENGELMAN, F. (1969). Female specific protein: biosynthesis controlled by corpus allatum in Leucophaea maderae. Science, N. Y. 165, 407-409. ENGELMAN, F. (1970). The Physiology of Insect Reproduction. New York: Pergamon Press. ENGELMAN, F. (1979). Insect vitellogenin: Identification, biosynthesis and role in vitellogenesis. Adv. Insect Physiol. 14, 49-108. ENGELS, W. (1972). Quantitative Untersuchungen zum Dotterprotein Haushalt der Honigbiene {Apis mellifica). Wilhelm Roux Arch. EnUoMech. Org. 171, 55-86. GELLISSEN, G., WAJC, E., COHEN, E., EMMERICH, H., APPLEBAUM, S. W. & FLOSSDORT, J.

(1976). Purification and properties of oocyte vitellin from the migratory locust. J. comp. Physiol. 108, 287-301.


15

F. (1979). In vitro induced pinocytotic activity by a juvenile hormone analogue in oocytes of Drosophila melanogaster. Cell Tiss. Res. 203, 241-247. GIORGI, F. (1980). Coated vesicles in the oocyte. In Coated Vesicles (ed. C. D. Ockleford & H. Whyte), pp. 135-177. Cambridge University Press. GIORGI, F. & JACOB, J. (1977). Recent findings on oogenesis of Drosophila melanogaster. III. Lysosomes and yolk platelets. J. Embryol. exp. Morph. 39, 45-57. GRABAR, P. & WILLIAMS, C. A. (1953). Methode permettant lYtude conjugee des proprietes 61ectrophoretiques et immunochimiques d'un milange de protdines: application au serum sanguin. Biochim. biophys. Acta 10, 193-215. HAGEDORN, H. H. & FALLON, A. M. (1973). Ovarian control of vitellogenin synthesis by the fat body in Aedes aegypti. Nature, Lond. 244, 103-105. HAGEDORN, H. H., FALLON, A. M. & LAUFER, H. (1973). Vitellogenin synthesis by the fat body of the mosquito Aedes aegypti. Evidence for transcriptional control. Devi Biol. 31, 285-294. HAGEDORN, H. H. & KUNKEL, J. G. (1979). Vitellogenin and vitellin in insects. A. Rev. Ent. 24, 475-505HANDLER, A. M. & POSTLETHWAIT, J. H. (1978). Regulation of vitellogenin synthesis in Drosophila by ecdysterone and juvenile hormone. J. exp. Zool. 206, 247-254. KAMBYSELLIS, M. P. (1977). Genetic and hormonal regulation of vitellogenesis in Drosophila. Am. Zool. 17, 535-549KOCH, P. (1964). In Vitro Kultur und Enrwicklungsphysiologische Ergebnisse an Embryonen der Stabheuschrecke Carausius morosus (Br.). Wilhelm Roux Arch. EntwMech. Org. 155, 549-593KOEPPE, J. & OFENGAND, J. (1976). Juvenile hormone induced biosynthesis of vitellogenin in Leucophaea maderae. Archs Biochem. Biophys. 173, 100-113. KUNKEL, J. G. & PAN, M. L. (1976). Selectivity of yolk protein uptake. Comparison of vitellogenins of two insects. J. Insect Physiol. 22, 809-818. LAEMMLI, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, Lond. 227, 680-685. LAURELL, C. B. (1966). Quantitative estimation of proteins by electrophoresis in agarose gel containing antibodies. Analyt. Biochem. 15, 45-52. LEMANSKY, L. F. & ALDOROTY, R. (1977). Role of acid phosphatase in the breakdown of yolk platelets in developing amphibian embryo. J. Morph. 153, 419—426. LOWRY, O. H., ROSEBROUGH, N. J., FARR, A. L. & RANDALL, R. J. (1951). Protein measurement with Folin phenol reagent. J. biol. Chem. 193, 265-275. LUNDQUIST, A. & EMANUELSSON, H. (1979). Membrane production and yolk degradation in the earlyflyembryo (Calliphora erythrocephala) Meig. An ultrastructural analysis.,?. Morph. 161, 53-78. MANS, R. L. & NOVELLI, D. G. (1961). Measurement of incorporation of radioactive amino acids into proteins by a filter-paper disk method. Archs Biochem. Biophys. 94, 48-63. OUCHTERLONY, O. (1968). Handbook of Immunodiffusion and Immunoelectrophoresis. Ann. Arbor Science Publishers. PAN, M. L. (1971). The synthesis of vitellogenin in cecropia silkworm. J. Insect Physiol. 17, 677-689. PAN, M. L. (1977). Juvenile hormone and vitellogenin synthesis in the cecropia silkworm. Biol. Bull. mar. biol. Lab., Woods Hole 153, 336-345. PAN, M. L., BELL, W. J. & TELFER, W. H. (1969). Vitellogenic blood protein synthesis by insect fat body. Science, N.Y. 165, 393-394. PAN, M. L. & WYATT, G. R. (1971). Juvenile hormone induces vitellogenin synthesis in the monarch butterfly. Science, N.Y. 174, 503-505. PFLUGFELDER, O. (1937). Bau, Entwicklung und Funktion der Corpora allata und cardica von Dixippus morosus (Br.). Z. iciss. Zool. 149, 477-512. POSTLETHWAIT, J. H. & WEISER, K. (1973). Vitellogenesis induced by juvenile hormone in the female sterile mutant apterous four in Drosophila melanogaster. Nature, Lond. 244, 282-285. REGIER, J. C. & KAFATOS, F. C. (1971). Microtechnique for determining the specific activity of radioactive intracellular leucine and applications to in vivo studies of protein synthesis. J. biol. Chem. 246, 6480-6488. ROTH, F. T., CUTTING, J. A. & ATLAS, S. B. (1976). Protein transport. A selective membrane mechanism. J. supramolec. struct. 4, 527-548. GIORGI,

16


H. (1977). Endocrine control of oogenesis in the housefly Musca domestica vidna. J. Insect Physiol. 23, 1295-1302. WALLACE, R. A. (1978). Oocyte growth in non mammalian vertebrates. In Tlie Vertebrate Ovary (ed. R. E. Jones), pp. 469-502. New York: Plenum Publishing. WALLACE, R. A. & JARED, D. W. (1976). Protein incorporation by isolated amphibian oocytes. V. Specificity for vitellogenin incorporation. J. Cell Biol. 69, 345-351. WALLACE, R. A. & HOLLINCER, T. G. (1979). Turnover of endogenous microinjected and sequestered proteins in Xenopus oocytes. Expl Cell Res. 119, 277-287. WYATT, G. R. & PAN, M. L. (1978). Insect plasma proteins. A. Rev. Biochem. 47, 779-817. SAKURAI,

{Received 18 February 1980 - Revised 27 May 1980)