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 MACCHIIstituto di Istologia e Embriologia, Universitd di Pisa

SUMMARY

Vitellogenesis in the stick insect Carausius morosus (Br.) has been studied with the goal ofidentifying 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 ofinjection. Incorporation of radioactivity under these conditions is shown to depend uponaccumulation of proteins rather than on a differential rate of protein synthesis in succeedingstages -of oogenesis.

By immunochemical analyses, it is shown that at least two antigens are common to bothhaemolymph and ovary and that one of these is also present in the fat body. Both antigens arelabelled during exposure to radioactive amino acids. When analysed by SDS polyacrylamidegel electrophoresis, extracts from both haemolymph and ovary appear to share a number ofprotein fractions which range in molecular weight from 40000 to 200000 Daltons. The labellingpattern exhibited by these fractions is clearly indicativ e of a protein transfer from the fat body tothe oocyte. Fat body cultured in vitro for up to 4 h releases a major macromolecular complexin the external medium. The latter has been identified as vitellogenin by both immuno-precipitation assay and SDS polyacrylamide gel electrophoresis. The protein which is syn-thesized and secreted under these conditions results from the processing of a protein complexof higher molecular weight.

INTRODUCTION

Vitellogenesis is a key event in ovarian development. It occurs by synthesis of aspecific yolk precursor, vitellogenin, in a tissue other than the ovary, namely, the fatbody (Brookes, 1969; Pan, 1971). Vitellogenin is not stored to any extent but is rathersecreted into the maternal haemolymph soon after being synthesized by the producingtissue (Pan, Bell & Telfer, 1969). Turnover of vitellogenin in the haemolymph isthought to result from a balanced equilibrium between the rate of secretion by thefat 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 amountsof vitellogenin and store it in a molecular form made insoluble by a mechanism as yetnot fully understood (Bergink & Wallace, 1974; Giorgi & Jacob, 1977). Apparently, itlooks as though vitellogenin may escape degradation only when taken into the oocyteby pinocytosis (Giorgi, 1980) and stored in the socalled yolk spheres (Wallace &Hollinger, 1979). Injected vitellogenin is, in fact, degraded by the oocyte in a mannernot dissimilar from that of other serum proteins (Dehn & Wallace, 1973). Con-

Author'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 madeavailable to the developing embryo after fertilization, only if maintained in amembrane-bound cell compartment (Lemansky & Aldoroty, 1977; Lundquist &Emanuelsson, 1979).

In order for an oocyte to accomplish vitellogenesis, several basic conditions haveto be fulfilled. First, vitellogenin titre in the haemolymph has to be sufficiently highto sustain vitellogenic maturation of all developing oocytes. Secondly, the oocyteitself 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 eachother so as to keep ovarian development in balance with the animal's general nourish-ment (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 anduptake of vitellogenin are both triggered by availability of juvenile hormone in thehaemolymph 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 tomost of the oviparous species with good approximation, including non-mammalianvertebrates (Wallace, 1978), there seems to be an increasing demand for extendingour present knowledge to species other than those which are currently used as modelsfor developmental biology. Based on these considerations we thought it of interest toextend our investigation on vitellogenesis to a parthenogenetic species, as is the casefor Carausius morosus, for which such aspects as sex determination and genetic controlof 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 byseveral tissues. On the basis of the criteria outlined above, this study aims to identifyvitellogenin, its precursor(s), if any, and its processed products in these tissues. It isthus intended to serve as a possible basis for purification and further biochemicalanalysis of the vitellogenin molecule.

MATERIAL AND METHODS

Specimens of the parthenogenetic species Carausius morosus (Br.) were reared in perspexcages and maintained on a diet of fresh ivy leaves. Females of similar age and showing acomparable egg producing rate were used throughout this experimental work. Prior to dissec-tion, animals were anaesthetized in a gaseous flow of CO|. Haemolymph was drawn from theanimal by using a sterile plastic syringe. To prevent haemolymph clotting, which is known tooccur upon exposure to air (Brehelin, 1979), haemolymph was diluted with 1 vol. of 1 mMEDTA in 10 % glucose solution. Ovarioles were removed from the abdominal cavity while theanimal was maintained in physiological saline solution. They were then transferred to a smallplastic Petri dish, divested of their enveloping layers and measured under a stereo-microscopeusing a calibrated eye piece. The oocyte volume at each developmental stage was calculated onthe assumption that the oocyte corresponds to a rotational ellipsoid. Accordingly, only the 2major axes of each oocyte were measured. Following complete removal of all ovariole andintestine, 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 homogenizationin 0-05 M Tris-HCl buffer at pH 6-8. Tissue extracts were stored in small aliquots at — 20 °Cuntil 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 byinjecting, subcutaneously and intramuscularly, 30 mg of antigen emulsified with an equalamount of complete Freund's adjuvant. Following a second injection of antigen emulsifiedwith incomplete Freund's adjuvant, the rabbits then underwent a weekly series of alternativebleedings and intravenous antigen injections. After blood clotting, rabbit antiserum was usedfor immunological analyses with no further purification. Tissue extracts were analysed againsttheir respective antisera by double immunodiffusion (Ouchterlony, 1968), by immunoelectro-phoresis 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 intooocytes was assessed by placing each oocyte, previously measured with a stereomicroscope, ina 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-precipitablematerial 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 withethanol-ether, washed again in ether and finally dried (Mans & Novelli, 1961). The paperdisks 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 aBeckman LS-100C counter.

In vitro culturing

Fat body was excised from the abdominal body wall as already described and cultured inGrace's medium (Gibco) for periods of 1, 2 or 4 h. Ten microCuries of fHJaspartic acid wereadded to 1 ml of culture medium supplemented with 50 fil of fresh haemolymph. All glasswareused in this experiment was made sterile by overnight exposure to an ultraviolet lamp andincubation media were sterile filtered. For each time point, aliquots of 100 fii of culture mediumwere incubated overnight with 100 pX of anti-egg antiserum. To check for specificity of thereaction, the immunoprecipitate was first washed in 005 M Tris-HCl buffer at pH 6'8 and thendissolved in 2 % sodium, dodecyl sulphate (SDS) solution in the same buffer. Finally, it wasanalysed by 10% SDS-polyacrylamide gel electrophoresis and processed for fluorography asdescribed below.

Electrophoresis and fluorography

10 % SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was carried out in a verticalslab gel apparatus according to the procedure of Laemmli (1970). Tissue extracts were dilutedwith an equal volume of SDS sample buffer (2 % SDS in 0-05 M Tris-HCl buffer at pH 6-8containing 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 determinedby comparison with standard proteins of known molecular weight which were run in the samegel. 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 aplot of molecular weights versus their relative electrophoretic mobility was made. Upontermination of electrophoresis, polyacrylamide gels were stained overnight in 1 % Coomassiebrilliant blue G250 in 45 % ethanol-10 % acetic acid and destained through several changes in

4 F. Giorgi and F. Macchi

the same solution. Since sample proteins were electrophoresed in duplicate for each exposuretime, 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 exposedin light-proof boxes for periods of time ranging from one to several weeks. Fluorography wascarried out by dehydrating half of each slab gel with dimethyJsulphoxide (DMSO) followed byimpregnation in 22-2 % PPO solution (w/v) in DMSO. Once dried as above, the slab gels wereexposed 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, wefirst 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 alldevelopmental 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 involume - appear to become far more active in incorporating radioactive amino acidsthan those of either larger or smaller sizes. We also noted that when oocytes of thesesizes are exposed in vivo for periods of time longer than 6 h, they appear to incorporateradioactivity at proportionally higher rates. In addition, as exposure in vivo is pro-longed from 12 to 24 h, the volume at which oocytes exhibit the highest radioactivityincorporation becomes progressively larger. These observations imply that under ourexperimental conditions, incorporation into medium-size oocytes is mainly due tolabelling of proteins which are not subjected to turnover. Whether this pattern oflabelling is due to ovarian-synthesized proteins or to proteins taken into the oocytefrom 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 periodof in vivo exposure to radioactive amino acids. Incorporation of [3H]aspartic acid intoTCA-precipitable proteins from oocyte extracts rises linearly over the time periodtested (Fig. 2 c). By comparison, pH]aspartic acid incorporation into fat body andhaemolymph proteins appears to follow a steady rise for up to 24 h and then todecline rapidly afterwards (Fig. 2 A, B). While the pattern of labelling observed inovarian tissues is clearly indicative of a steady accumulation of the radiolabelledproteins, 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 aminoacid pool or rather to the fact that the protein(s) synthesized during the period ofexposure are exported outside the tissues themselves. Although total counts in thehaemolymph are still very high at the time protein labelling appears to decline, thefirst hypothesis cannot be completely ruled out. This could be done only by knowingthe exact extent of the intracellular amino acid pool (Regier & Kafatos, 1971).

To pursue further the idea of protein transport from one compartment to anotherof the vitellogenic system, we attempted to examine whether the selected tissuesshare a common protein content among themselves. As shown in Fig. 3 A, there are atleast 2 major antigens which are common to both oocytes and haemolymph whenextracts of these tissues are made to react with their respective antisera. When such a

Vitellogenesis in Carausius 5

comparison is extended to fat body extracts, it appears that at least 1 of the 2 antigensshared by oocytes and haemolymph is also present in this tissue (Fig. 3B). A similarconclusion could be drawn from the immunoelectrophoretic analysis shown inFig. 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., mm3

Fig. 1. Extent of amino acid incorporation as a function of oocyte volume. Oocyteswere 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 incorporationexpressed as total counts over time. The ratio between incorporation of PH]asparticacid to ["SJmethionine is also indicated ( ).

Fig.

6 12 24 6 12 24 48

Exposure time, h

6 12 24 48

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

F. Giorgi and F. Macchi

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 immunoelectrophoresison agarose plates containing 1/10 of anti-egg antiserum. Samples of haemolymphwere exposed in vivo to [•)5S]methionine for 3,6, 12, 18, 24 or 48 h and then electro-phoresed 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 wereexposed in vivo for 6 h to [3°S]methionine. Electrophoresis in the first dimension wasperformed at 200 V/ 25 mA for 30 min; subsequently, the antigens were left todiffuse overnight against their respective antisera. E, F, autoradiographs of the platesshown in C and D.

Vitellogenesis in Carausius y

oocytes and haemolymph are better evidenced by analysing aliquots of these tissuesby rocket immunoelectrophoresis (Fig. 3 c). Both these antigens, though variable intheir 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 theirantigenic composition with each other and that protein labelling occurs at a rate whichis sufficiently high to be studied during the periods selected for in vivo exposure, wewere 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 proteinfractions revealed by SDS polyacrylamide gel electrophoresis in oocytes, only 6 exhibitan electrophoretic mobility similar to that of the fractions present in the haemolymph(Fig. 4A). Comparison with fat body electropherogram is made difficult by the largenumber of protein fractions which are detectable by this technique and by the lowrepresentation of each fraction in this tissue. Since standard proteins of knownmolecular weight were electrophoresed along with those of the vitellogenic tissues,their shared protein fractions are, hereafter, termed by making reference to theirestimated molecular weight (Fig. 4 A).

The relative rates of protein synthesis in these tissues can be qualitatively estimatedby comparing the set of fluorographs shown in Fig. 4B. After 1 h of exposure toPHJaspartic acid, the protein fractions which exhibit the highest specific activity inthe fat body are those of 200000 and 250000 molecular weight. A protein fractionshowing an apparently similar or slightly smaller molecular weight than 200000,although with less amount of label on it, is also present in the haemolymph. Noprotein fraction is labelled in the oocyte at this time interval. Other major proteinfractions which become labelled in the haemolymph at this exposure time are theones with molecular weights around 100 000, 125000, and 130000. The last of theseprotein fractions has also a counterpart in the fat body, though much less labelledthan in the haemolymph. With progressively longer exposures to radioactive aminoacids, the pattern of labelling in each tissue changes drastically. The essential featureof these patterns is the appearance of the 200000 protein fraction in the oocyte. Thisis also the only protein fraction to exhibit an increasing amount of label as timegoes by.

When fat body is exposed for 24 h in vivo to radioactive amino acids and thenchased for up to 4 h in an in vitro system, the amount of radioactivity appearing in themedium in the form of proteins precipitable by the anti-egg antiserum increasesslightly over the same time period (Fig. 5 A). A somewhat similar release of antiserum-precipitable protein could be obtained when fat body, not previously exposed toradioactive amino acids, was incubated in vitro in a radioactive medium (Fig. 5B).However, under these conditions the radioactivity incorporated into fat body proteinsdoes not decline, but follows a pattern of incorporation more readily comparable tothat detectable during the first phases of in vivo exposure (see Fig. 2 A).

Analysis by SDS-polyacrylamide gel electrophoresis of the proteins immuno-precipitated from the culture medium revealed that at least 60% of the total precipitate

F. Giorgi and F. Macchi

A300 K.

165 K •

1 5 5 K '

68 K

43 K

39 K

-as.

a 4 250 K

b « 200 K

c 4 125 K• d « 100 K

e 85 K

. f 4 60 K• g ^ 55 K

h 50 K

• i 40 K

21-5 K

B

ir ii

I

Vitellogenesis in Carausius 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 theprotein released by the fat body in vitro is indeed the same fraction which is transferredto the maternal haemolymph and to the oocyte (Fig. 6 A).

To determine whether the protein fraction released by the fat body in the culturemedium results from processing of a larger polypeptide, we again cultured fragmentsof this tissue in a radioactive medium and then analysed their SDS-solubilized extractsby gel electrophoresis. The results of this experiment indicate that the 2 major fractionswhich are synthesized by the fat body under these conditions, are labelled in a se-quential order (Fig. 7). In fact, as label decreases in the heavier fraction, it decreasesin 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 eitherin vivo (A) or in vitro (B) exposure to PHJaspartic acid. Proteins released in the mediumunder these culturing conditions were immunoprecipitated with anti-egg antiserumand the resulting radioactivity expressed as cpm over mg of fat body protein and100 fi\ of culture medium. Labelled proteins retained by the fat body during in vitroculturing (O—O; • — • ) and labelled proteins released in the culture mediumover 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 molecularweights (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), trypsininhibitor, 21500 (Boehring). Protein fractions shared by ovary and haemolymph aremarked (^) . B, electrophoretic analysis (10% SDS-PAGE) of the proteins syn-thesized by fat body (b), haemolymph (e) and ovary (ov). Samples were electro-phoresed 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 F. Giorgi and F. Macchi

DISCUSSION

Vitellogenin has been traditionally defined as the sex-specific protein in the haemo-lymph which comprises much of the yolk proteins in the mature egg (Hagedorn &Kunkel, 1979). This definition has been questioned on the grounds that otherhaemolymph proteins may as well gain access to the oocyte during vitellogenesis(Engelman, 1970, 1979). In spite of this criticism, however, the term vitellogenin canstill be usefully employed, provided the rate of uptake of this protein into the oocyte iscarefully measured and so compared with that of other oocyte proteins having anexogenous 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 fromovarian extracts, and B, fluorograph of the fat body protein released under in vitroculturing 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 lastconsideration 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 notentirely satisfactory.

V

•

* A

* B

c

Fig. 7. Densitometric scans of fluorographs of the proteins synthesized by fat bodyunder in vitro conditions following exposure to ["HJaspartic acid for 1 (A), 2 (B), and4 h (c). Large arrow indicates origin, small arrows indicate the 2 protein fractionswhich are labelled sequentially over the period tested.

This study reports on a first attempt to identify vitellogenin in the haemolymph ofCarausius morosus. The criteria we have set forth in this study are based on a variety ofexperimental approaches all of which have to be considered as preliminary in view ofthe final check on selectivity. Our data show that oocytes exposed to radioactiveamino acids for various lengths of time become maximally labelled in the volume range2-5 mm3 with a minimum delay of 6 h after the time of injection. Similar observationshave previously been carried out in oocytes of other insects by either autoradiographic

12 F. Giorgi and F. Macchi

(Bier, 1962, 1963) or electrophoretic techniques (Engels, 1972). All these studies areconsonant with the view that oocyte labelling is primarily due to proteins synthesizedby a tissue outside the ovary and then transferred to the oocyte only after secretioninto the haemolymph. The time lapse required by the oocyte to become maximallylabelled can, in fact, be reasonably attributed to the time required by an extraovariantissue to synthesize and secrete this protein and to that necessary for its transfer to theoocyte. That such a scheme could also be applied with reasonable confidence to oocytelabelling in Carausius morosus is confirmed by the observation that incorporation ofradioactivity into oocytes does not turnover during the time period examined.Stability of the proteins is, in fact, a necessary though not sufficient criterion by whichone can identify vitellogenin in ovarian extracts. The data on the labelling pattern ofthe TCA-precipitable material has in addition shown that the ovary is the only tissueof those examined to exhibit a steady rate of accumulation of radioactivity. The lastconsideration is also consistent with the interpretation that labelling of progressivelylarger oocytes, as it occurs with prolonged exposure to radioactive amino acids, islikely to depend upon accumulation of proteins rather than on a differential rate ofprotein synthesis in succeeding stages of vitellogenesis.

To elucidate the nature of the material accumulated in oocytes, fat body andhaemolymph from adult females were analysed as well. By the use of several immuno-logical techniques we have been able to show that at least 2 antigens are common toboth ovary and haemolymph and that one of these is present in the fat body. Althougha common antigenic content among these tissues does not prove per se the existence ofprotein 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 wayof release into the haemolymph has been obtained by the use of fluorographic andelectrophoretic techniques. We have shown that each of the tissues examined by thesetechniques contains a predominant protein fraction with an estimated molecular weightof about 200000 which, during in vivo exposure to radioactive amino acids, is labelledin a sequential order: from fat body to oocyte.

As a final experiment in our present series we have shown that the major proteinfraction which is released both in vivo and in vitro by the fat body has a molecularweight which falls well within the range of that estimated for the heavily labelledfractions of the haemolymph and ovary. On the basis of the evidence so far gatheredwe feel confident in identifying the 200000 molecular weight protein fraction as thevitellogenin 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 vitello-genin identification. The first of these concerns the relationship between the proteinfraction resolved by gel electrophoresis under denaturing conditions and its nativeform. The second is whether the 2 antigenic forms, which are detected in the haemo-lymph by immunoelectrophoresis against anti-egg antiserum, may both be identifiedas 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 inits native form has a molecular weight of around 5 x io6 Daltons in most of the insectspecies so far examined (Wyatt & Pan, 1978). In addition, investigations of vitellogenin

Vitellogenesis in Carausius 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 havebeen detected by SDS polyacrylamide gel electrophoresis in the vitellogenin moleculeisolated from Cecropia pupal haemolymph (Kunkel & Pan, 1976). A similar subunitcomposition has been found for the vitellogenin of Philosomia cynthia, althoughhigher molecular weights were reported for its subunits (Chino, Yamagata & Sato,1977). A somewhat different situation has been reported in cockroaches and in themigratory 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 applicabilityof the scheme outlined above for the vitellogenin in Carausius, although as yet nocertainty has been gained as to the exact number of subunits in this species. Whether1 or 2 vitellogenin peptides exist in the native state in Carausius cannot be decided onthe basis of the evidence provided by this study. Although instances of more than onevitellogenin have occasionally been reported in the literature (Hagedorn & Kunkel,1979), we believe measurement of the relative rate of oocyte uptake for each of thehaemolymph antigens is the most reliable means of solving this question. At thepresent time, we are inclined to believe that a unique form of vitellogenin exists inCarausius; this belief is based upon the observation that 1 haemolymph antigen is byfar the most abundantly represented in ovarian extracts.

In the final part of our study we demonstrated that fat body taken from adultfemales in Carausius is capable of releasing a labelled macromolecular complex intothe external medium. Such a complex has been identified as vitellogenin by bothimmunoprecipitation assay and SDS-electrophoresis. The vitellogenin released underthese conditions amounts to about 60% of the total protein found in the medium after4-h culturing, implying that other haemolymph proteins may be secreted as well bythe fat body during the same period of time. Similar or even higher rates of vitellogeninsecretion have been previously reported for other insect species (Chen, Couble,Abu-Hakima & Wyatt, 1979). In parallel with these findings, we have also providedsome evidence that the vitellogenin secreted by fat body results from a proteolytictrimming of an intracellular precursor of higher molecular weight. The evidenceagrees with earlier observations in other insects (Koeppe & Ofengand, 1976; Chen,Strahlendorf & Wyatt, 1978). The processing of vitellogenin in the fat body is likelyto occur prior to secretion and could thus constitute a necessary step in the release ofthe partially degraded product. In spite of the present incompleteness of ourknowledge of the intracellular processing for secretory proteins, the evidence providedin this study, along with that by previous investigators, points to the general occurrenceof such a phenomenon.

Whether vitellogenin synthesis in Carausius is under hormonal control as in manyother insects (Doane, 1973) or rather occurs independently of a hormonal supply as inCecropia (Pan, 1977) could not be established beyond doubt in this study. However,our preliminary observations on isolated abdomens from adult females would seem toindicate that both synthesis and uptake of vitellogenin in Carausius occur irrespectiveof a hormonal control. Earlier evidence by Pflugfelder (1937) that removal of corpora

14 F. Giorgi and F. Macchi

allata in adult Carausius has apparently no effect on vitellogenesis is also consistentwith this interpretation. Perhaps it is the occurrence of continuous egg productionthat makes unnecessary the existence of an endocrine regulation for vitellogeninsynthesis and uptake in the stick insect.

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{Received 18 February 1980 - Revised 27 May 1980)