Isolation and quantification of vitellogenin in the haemolymph of the ovoviviparous cockroach Nauphoeta cinerea

Comp. Biochem. Physiol. Vol. 76B, No. 1, pp. 65 to 72, 1983

0305-0491/83 $3.00+0.00 (c~ 1983 Pergamon Press Ltd

Printed in Great Britain

ISOLATION A N D QUANTIFICATION OF VITELLOGENIN IN THE HAEMOLYMPH OF THE OVOVIVIPAROUS COCKROACH NA UPHOETA CINEREA J. BUSCHOR and B. LANZREIN Department of Animal Physiology, University of Berne, Erlachstrasse 9a, 3012 Berne, Switzerland (Tel: 031-65-83-49)

(Received 31 January 1983) Abstract--l. Warming the insects for 2 rain at 57°C prevents haemolymph from clotting and yields high amounts of unchanged vitellogenin. 2. Purification on Sephadex G-25 and DEAE did not alter vitellogenin as concluded from native PAGE and immunodiffusion. 3. Measurement of vitellogenin concentration in haemolymph by rocket immunoelectrophoresis revealed a great variation among individual females at the same stage. 4. During an oocyte maturation cycle vitellogenin increases until ovulation (32 mg/ml), when it makes up 58% of the total haemolymph proteins. After ovulation the vitellogenin titer decreases and remains low during pregnancy.

MATERIALS AND METHODS

INTRODUCTION

Insects Nauphoeta cinerea were kept at 26°C and 60~/or.h. on dog

In ovoviviparous cockroaches such as Nauphoeta cinerea the sexual cycle of the female is divided into oocyte maturation and gestation. During the oocyte maturation period only the terminal oocytes grow and juvenile hormone (JH) is the principal agent controlling events such as vitellogenin (VG) synthesis in the fat body and uptake of V G into the oocytes (for a review see Engelmann, 1979; Lanzrein et al., 1981). The term V G describes yolk protein precursors produced by the fat body and released into the haemolymph while the term vitellin is used for the yolk proteins within the oocyte. V G from several species has been purified and characterised (reviewed by Wyatt and Pan, 1978; Hagedorn and Kunkel, 1979) and in many insects, including Nauphoeta cinerea, VG and vitellin are female-specific proteins not normally present in the male (Bfihlmann, 1974; Englemann, 1979). We have observed in this insect that corpora allata activity in vitro and in vivo and haemolymph J H titer are very different and not correlated in individual females of the same age and stage (Lanzrein et al., 1978). In order to investigate a role of V G in regulating corpora allata activity and mechanisms of V G release and uptake it was thus necessary to develop techniques for isolating and purifying VG as well as for quantifying it in individual females. In the present study we report on ways of collecting haemolymph whereby clotting can be minimised and on methods for purifying V G and quantifying it in the haemolymph of individual females. We present measurements of total protein content and VG concentration in haemolymph during the first oocyte maturation cycle and in early pregnancy and compare our data with those obtained in other ovoviviparous cockroaches as well as with oviparous and viviparous species.

flakes and water at a photoperiod of 12 hr. Under these conditions oocyte maturation was 12-13 days and gestation 3540 days. Ovariectomy was performed 15-20hr after adult emergence through incisions in the last sternite.

Collection of haemolymph, chromatography

76/1

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DEAE-

In order to collect the haemolymph, insects anaesthetised with CO 2 were submerged in a water-bath at 57°C for 2 rain (see Table 1) and bled by cutting one leg and squeezing the abdomen and thorax. The haemolymph was then centrifuged for l0 rain at 600g in Gilson disposable tips welded on the pointed side. For VG quantification the supernatant was either immediately used in rocket immunoelectrophoresis or stored at -20°C. Hereafter this supernatant is referred to as haemolymph. For VG isolation the haemolymph was filtered through glass-wool to remove the lipid-containing upper layer. In order to remove low molecular weight substances and to dissolve the haemolymph in an appropriate buffer the material was then either run over a gel filtration column (9 × 1.5cm) of Sephadex G-25 (Medium, Pharmacia), equilibrated with DEAE starting buffer (see below) or dialysed against DEAE starting buffer. The material corresponding to 1 ml of haemolymph was then applied at 4°C to a column (1.6 × 12cm) of DEAE Sepharose CL-6B (Pharmacia), equilibrated with starting buffer (0.02M phosphate, 0.15 M NaC1, 0.02% NaN 3 at pH 7.4, 7.0 or 6.5; see Results). The flow rate was 28 ml/hr and 3 ml fractions were collected. After washing with the same buffer until the effluent gave A280nm< 0.02, VG was eluted using a gradient of increasing NaCI (0.15~0.6M). Fractions were examined with an Ouchterlony immunodiffusion test (Ouchterlony, 1967) using a VG specific antiserum.

Affinity ehromatography CNBr-activated Sepharose 4B (Pharmacia) was used and ligands were coupled to the matrix according to the Pharmacia handbook (affinity chromatography). The columns were 65

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gel filtration,

66

J. BUSCHOR and B. LANZREIN Table 1. Quantity of haemolymph and VG obtained from 8-day-old females under various conditions

Time (min) 2 2 2 2 1 2 4 2

Conditions Temperature (~C) 53 57 61 69 57 57 57 20

Insects with clotting haemolymph (~o)

Amount of haemolymph collected (#l/insect)

VG (mg/ml)

60 0 0

10.2 _+9.0 22.0 _+6.7 19.7 _+ 5.9 <2 ~1 10.2 _ 10.6 22.0 _+ 6.7 20.3 + 8.6 < 2/~ 1

8.6 + 5.0* 10.0 _+ 8.2 13.4 _+ 5.8 not measurable 11.6 _+4.5? 10.0 _+ 8.2 11.4 +_ 8.8 not measurable

50 0 0 100

Mean of 10 individual insects _+ SD; except for (*) and (t), where only the 4 and 5 non-clotting samples respectively were analysed. equilibrated in 0.1 M phosphate, 0.5 M NaC1, 0.02~o NaN 3, pH 7.0. Samples were applied at a flow rate of 5 ml/hr and bound proteins were eluted with 3 M NaSCN pH7.5 (v = 12 ml/hr) and immediately dialysed against a phosphate buffer. Immersible CX ultrafilters (Millipore) were used to concentrate the effluent fractions.

Polyacrylamide gel electrophoresis (PAGE) Native slab gels (1.5mm, 14 x 7cm) containing 7.5}~, polyacrylamide were prepared according to Maurer (1971). Each sample contained 5-50 #g protein in 30/~1 of Tris-HCl buffer (0.2 M Tris, 0.1 M NaC1, pH 6.7) containing 20~o glycerol and 0.001~o bromophenol blue. Electrophoresis was carried out at room temperature in a Tris/glycine buffer at pH 8.3 (Maurer, 1971) at a constant current of 30 mA for 3 hr. Gels were fixed in 10~o TCA and stained for proteins in a solution of methanol:HzO:acetic acid (5:5:1) containing 0.25~ Coomassie Blue R 250 (Merck). Native disc gel electrophoresis was carried out according to Maurer (1971). Separating gels were 9cm long with a diameter of 6 mm and with a linear gradient of 3.8-12.4~ acrylamide. The spacer gels contained 3.1~ acrylamide and were 400 #1 in volume. Electrophoresis was carried out at room temperature at a constant current of 1.7 mA per gel in a Tris/glycine buffer (see Keleti and Lederer, 1974). Gels were fixed and stained as described above. Details of the method will be published later (Imboden, in preparation).

Preparation of antiserum, lipoprotein-immunoelectrophoresis An antiserum against female haemolymph prepared by Biihlmann (see Biihlmann, 1976) was rendered femalespecific by passage over a Sepharose 4B affinity column to which male haemolymph proteins were coupled. By this method a VG-specific antibody is obtained, as can be seen in Ouchterlony immunodiffusion, where female haemolymph and yolk homogenate form a single fused precipitation line (Bfihlmann, 1974). This antiserum was used for Ouchterlony and rocket immunoelectrophoresis. For affinity chromatography and immunoelectrophoresis 1 mg of DEAE-purified VG, emulsified with Freund's complete adjuvant (Difco), was injected i.m. into rabbits four times at intervals of 2 weeks. The antiserum was precipitated with 45~ (NH4)2SO 4 and redissolved in equilibration buffer. The IgG were gained by protein A-Sepharose chromatography and 0.1 M glycine/HC1, pH 3.0 as an elution buffer. The lgG were then run over two affinity chromatography columns, the first with male haemolymph proteins and the second with DEAE purified VG as ligands. The specific elution of the second column gave a pure fraction of anti-VG IgG. Quantification of electrophoretically separated lipoproteins is possible by a chemical precipitation with polyanions using Rapidophor (Immuno-Diagnostica GmbH, Mannheim), a ready-to-use system normally used for quan-

tifying human plasma lipoproteins. The precipitations were measured densitometrically and compared to the immunoprecipitations.

Quantification of VG and total proteins DEAE purified VG (about 2 mg/ml) was dialysed against 0.1 M NaC1, 0.02% NaN 3 since the VG of Nauphoeta cinerea is not soluble in lower salt concentrations. The sample was lyophilised and weighed after both the A280,mand the precise volume had been determined. The relationship between A280nm and VG concentration (mg/ml) was calculated taking into account the weight of NaC1 and NaN 3. VG concentrations were estimated by rocket immunoelectrophoresis according to Laurell (1966). Antiserum containing agarose (Serva 11401) was poured on to a glass plate (8 x 10cm). Samples (5lzl) were always applied together with three dilutions of DEAE purified standard VG. Fob lowing Svendsen's method (see Axelsen et al., 1973), a barbital-glycine/Tris buffer with pH 8.8, having a high buffer capacity and a low ionic strength, was used in a dilution of 1:2. The electrophoresis was run for 15hr at 3 V/era in a water-cooled chamber (LKB 2117 Multiphor). The height of the rockets was measured after washing with saline and H20 and staining with 0.5~o Coomassie Blue R 250 in H20:acetic acid:ethanol = 9:2:9, and compared to the rockets of the VG standards. The protein content of the haemolymph was measured by Bradford's (1976) method using bovine serum albumin (MILES) as a standard.

RESULTS

Conditions Jor the collection of non-clotting haemolymph Since it was our goal to measure V G concentrations in individual females we h a d to work out conditions which would allow collection o f sufficient quantities (approx 5 # 1) of non-clotting h a e m o l y m p h . W e observed that cooling to 4"C was not suitable a n d then used high temperatures to prevent clotting. Females were w a r m e d in a w a t e r - b a t h at various temperatures a n d for various lengths of time (Table 1). W a r m i n g the insects for 2 min at 57°C was found to be most suitable, since it prevented clotting a n d a high a m o u n t of h a e m o l y m p h could be o b t a i n e d ( a b o u t 10~o of the total h a e m o l y m p h ) . F r o m this point on we always applied these conditions. W a r m ing to a lower t e m p e r a t u r e (53°C) or for a shorter time (1 min) resulted in an inconsistent reaction from no clotting to total clotting, while w a r m i n g for a

Vitellogenin in Nauphoeta c&erea

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tion of VG, and obviously its migration velocity is the same under the two bleeding conditions.

Preparation of VG

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For preparation of VG, female cockroaches were ovariectomised 15 hr after adult ecdysis and kept for 28-30 days, which results in a massive accumulation of VG (Lanzrein et al., 1981). In order to get an idea about the relative abundance of VG and high molecular weight substances (mol.wt> 5000daltons) in haemolymph of oocyte maturing and ovariectomised females, the A280nm was determined for total haemolymph, for haemolymph after gel filtration on Sephadex G-25, and for VG, where rocket immunoelectrophoresis using a VG reference of known m280nm was applied (Table 2). One can see that both VG and total protein content are remarkably higher in ovariectomised females (VG 4.4 times, total protein 1.8 times higher) than in normal females. Low molecular weight substances make up about 27-33~ of the A2s0nm in haemolymph, a finding also confirmed by dialysis (data not presented). VG can be separated from other haemolymph proteins using DEAE column chromatography under the appropriate conditions (Fig. 3). At a pH of 7.4 the VG containing peak is eluted at 0.27 M NaC1 (A); lowering the pH to 7.0 and 6.5 (B, C) revealed an additional peak containing no VG, as tested by Ouchterlony immunodiffusion; pH 6.5 (0.02 M phosphate) thus became our routine system where VG eluted in a single symmetrical peak at 0.33 M NaCI (C). The VG peak is missing in male haemolymph

A

B

C

D

Fig. 1. Ouchterlony immunodiffusion test of haemolymph and purified VG with antiserum against cold-bled femalespecific proteins (A). (a) 1. 10-day-old females, bled after 2 rain at 57°C, (diluted 1:32). 2. DEAE purified VG. 3. 10-day-old females, bled after 3 hr at 4°C (diluted 1:2). 4. 10-day-old females, bled after injection of 100pl of insect Ringer solution and cooled for 1 hr at 4°C (diluted 1:4). 5. 10-day-old males, bled after 2 min at 57°C (diluted 1:4). 6. VG after purification by DEAE and affinity chromatography. (b) 7. Same as 6, but using a higher concentration.

longer time (4 min) or at a higher temperature (61°C) gave results similar to those obtained by warming for 2 min at 57°C, but certainly these conditions are less favourable for proteins. After warming to 69°C no haemolymph could be collected, probably because too much protein was denatured. In order to test whether warming the insects alters the VG, haemolymph bled under different conditions was analysed by Ouchterlony immunodiffusion (Fig. 1) and PAGE (Fig. 2). Figure 1 shows that adjacent wells containing female haemolymph obtained under various conditions (i, 3, 4) produce a single fused precipitation line with female-specific antiserum, thus indicating that warming does not alter the VG immunologically. The gels in Fig. 2 show a great similarity in the disc electrophoretic pattern from warm (A, C) and cold (B, D) bled haemolymph. Comparing the female with the male haemolymph reveals the posi-

Fig. 2. PAGE (discs with a linear gradient of 3.8-12.4~o acrylamide) of I 1-day-old female (A, B) and l l-day-old male (C, D) haemolymph. Haemolymph was collected either after warming the insects for 2 min at 57°C (A, C) or after cooling the insects for 60 min at 4°C and injecting (after 30 min) 150-200/21 of insect Ringer (B, D). Gels prepared by courtesy of Dr H. Imboden.

68

J. BUSCHORand B. LANZREIN Table 2. A280n m of l ml of total haemolymph, of haemolymph after gel filtration (mol.wt > 5000 daltons) and of the VG portion from 8-day-old females and 28- to 30-day-old females ovariectomised on day 0 Age A: Total haemolymph B: High molecular weight C: VG substances (~ proteins) 8 28-30 (ovariectomised)

86.8 + 5.3 = 100% 170.4_+5.4= 100%

63.4 _+2.3 = 73.0~o 117.2 -+ 8.9 = 68.8%

7.5 _+ 1.6 53.2_+6.8

Mean values 4-SEM from 4 to 5 samples of 30-80 females each (A and B), or l0 individual females (C). (D), indicating that it consists of female-specific proteins only. As a method of purifying VG, affinity chromatography turned out to be rather difficult. We tested different systems for eluting bound VG and found a complete recovery only by using 3 M NaSCN (pH 7.5). However, the immunodiffusion test (Fig. l b) revealed 2-3 precipitation lines, and also analysis using PAGE gave several bands (Fig. 4: G, H), indicating that VG had disintegrated and formed aggregates.

Examination of the VG purification steps by PAGE and immunoelectrophoresis Analysis of female and male haemolymph and of

the VG purification steps using PAGE is shown in Fig. 4(A-K). Some material always remains on top of the separating gel. A comparison between (A) (haemolymph of oocyte-maturing female) and (1) (male haemolymph) reveals that female haemolymph contains a protein which is absent in the male (see also Fig. 2) and which comigrates with the major protein obtained after DEAE chromatography (D, E, F). This protein is VG, as also concluded from immunological analysis (Fig. 1). In order to verify further whether the isolation procedure, including DEAE chromatography, alters the VG molecule, lipoprotein-immunoelectrophoresis (Fig. 5) was carried out. In the lipoprotein-immunoelectrophoresis system P.6-

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Fig. 3. DEAE chromatography of prepurified (Sephadex G-25) haemolymph from: 8-day-old females (1200/~1) at pH 7.4 (A); 8-day-old females (1100/~1) at pH 7.0 (B); 8-day-old females (900/~1) at pH 6.5 (C); 8-day-old males (800 #1) at pH 6.5 (D). Dotted line: NaCI gradient. Fractions containing VG elute at 0.27-0.33 M NaCl.

Vitellogenin in Nauphoeta cinerea

69

Fig. 4. PAGE (7.5~o acrylamide slab gel) of female and male haemolymph and of VG after different purification steps. (A) Haemolymph from 10-day-old females; (B), (C) Same as A, after gel filtration; (D), (E), (F) Fractions containing VG after DEAE chromatography; (G), (H) Same as D, after anti-VG affinity chromatography; (I) Haemolymph from 10-day-old males; (J), (K) Standard proteins (thyroglobulin, ferritin, catalase, lactate dehydrogenase, BSA; Pharmacia electrophoresis calibration kit).

the purified VG migrates slightly faster than the non-purified VG (Fig. 5), an observation similar to that in immunoelectrophoresis (Buschor, unpublished). The lipoprotein quantification (polyanionprecipitation) reveals one peak for the purified VG and two peaks for the haemolymph, but only one reacts with anti-VG. These data (Figs 1, 4, and 5) suggest that the VG molecule might be slightly changed upon purification by DEAE chromatography but that these changes are only recognised in immunoelectrophoresis in agarose gels but not in our native PAGE or Ouchterlony immunodiffusion.

Quantification of VG in individual females Lyophilisation of DEAE purified VG (see Materials and Methods) gave a conversion factor of 1.6 for calculation from A280nm into mg/ml. A typical example of rocket immunoelectrophoresis is shown in Fig. 6. On each plate we applied three dilutions of standard VG (St-S3) together with the samples. One can see a great variation in VG concentration among the individual females of the same age and the same physiological stage, an observation that we made throughout the titer measurements. Concentrations of 50 #g VG/ml can be detected by this method, and since only 5 pl samples are used the sensitivity is 0.25 #g. A titer curve for VG in adult females during oocyte maturation and the early stages of pregnancy is shown in Fig. 7, together with the total protein content and the dry weight of the ovary. The VG and the total haemolymph protein concentration could be measured only after day 2 because 0 and 1-day-old females did not yield enough haemolymph for quantification with the tests that we use. Between days 5 and 10, when the oocytes grow rapidly and incorporate VG intensively, the VG titer remains on a similar level, indicating an equilibrium between VG release by the

fat body and uptake by the oocytes. The titer reaches its highest value on day 12, when the females ovulate. Thereafter it falls within only 3 hr to a very low level and remains low during pregnancy (<0.5mg/ml). The protein titer is high until day 8 and then, at the time of most intensive oocyte growth, it decreases significantly and displays similar fluctuations to those of the VG titer between day 9 and day 12. During

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Fig. 5. Lipoproteinelectrophoresis combined with immunoprecipitation (below) and densitogram of polyanion precipitation (above) using the Lipidophor system from (a) DEAE purified VG and (b) 11-day-old female haemolymph. Antiserum against DEAE purified VG was used.

70

J. BUSCHORand B. LANZREIN

Fig. 6. Rocket immunoelectrophoresis of haemolymph from individual 8-day-old females (1-9) and of DEAE purified VG standard (SI $3). Haemolymph was diluted 1:33.3 with electrophoresis buffer, and VG standards were applied in concentrations of 0.32, 0.64 and 0.80 mg/ml. The sample volume was always 5/~ 1.

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Fig. 7. Haemolymph protein content (dots) and VG titer (circles) together with the ovary dry weight (triangles) from females during the first oocyte maturation cycle and early pregnancy. Mean+ SEM. Each value is based on measurements from 9 to 10 individual females, except for protein measurements between day 9 of oocyte maturation and day 3 of gestation, where 18-20 individuals were measured. Abscissa: age of females in days.

pregnancy (data not shown) the protein content was found to increase to values comparable to young adult females, namely 116 _+ 11 mg/ml on day 21 and 128 _+ 7.6 mg/ml on day 31 of pregnancy. DISCUSSION

In the present study methods for preventing haemolymph clotting, for isolating VG and for quan-

tifying it in the haemolymph of individual females are described. Immediate clotting of the haemolymph upon collection, a major problem which for a long time impeded protein measurements in individual females, could be overcome by warming the insects for 2 min at 57c'C (Table 1). This procedure does not alter the VG immunologically (Fig. 1), and we found that VG of warm and cold-bled haemolymph behaves the same in native (Fig. 2) and SDS PAGE (K6nig, unpublished). It is interesting to note that the majority of the haemolymph proteins seem to be unaffected by warming to 57°C as concluded from native PAGE (Fig. 2). Warm-bleeding leads to a higher VG yield than cold-bleeding (4°C) (cf. the different dilutions in Fig. 1), where considerable quantities of VG are retained in the clot (Buschor, unpublished). Freezing and thawing as applied for Leucophaea maderae (Koeppe et al., 1980) is not applicable to Nauphoeta since no haemolymph can be collected under these conditions in most cases. Clotting proteins-c o a g u l o g e n ~ h a v e been analysed in Leucophaea maderae (Bohn et al., 1981) and in Locusta migratoria (Brehelin, 1979) but nothing is known about the clotting mechanisms in Nauphoeta cinerea. Injection of clotting inhibitors is not suitable for our purposes since precise measurement of VG concentrations would not be possible. DEAE chromatography using a gradient of NaC1 is an efficient method for isolating VG provided appropriate pH and buffer are used (Fig. 3). In Locusta migratoria Gellissen et al. (1976) used a phosphate buffer at pH 7.6 for isolating vitellin while Chinzei et al. (1981), testing several pH's, achieved complete separation of lipoprotein I from VG and vitellin only using a phosphate buffer at pH 6.3 but not at a higher pH. This finding is comparable to that in Nauphoeta where lowering the pH from 7.4 to 6.5 (Fig. 3) allowed resolution of an additional peak not reacting with anti-VG. Lowering the pH to 6.3 as in Locusta migratoria (Chinzei et al., 1981) was not possible since this caused precipitation of VG. Prepurification of VG by precipitation with a low salt concentration, a frequently used method, was omitted because VG of Nauphoeta does not completely precipitate nor redissolve under these conditions. Our DEAE purified VG was found to be immunologically identical to haemolymph VG (Fig. 1) and the two show a similar behaviour in native PAGE (Fig. 4), but a slightly different migration velocity in immunoelectrophoresis using agarose gels (Fig. 5). This indicates that upon DEAE purification the VG molecule undergoes slight changes which become recognisable only in immunoelectrophoresis but not in immunodiffusion or in native PAGE. For routine quantification of VG in haemolymph of individual females at the vitellogenic stage rocket immunoelectrophoresis is a useful and reproducible method (Figs 6 and 7). We have recently developed an enzyme-linked immunosorbent assay (ELISA) for measuring very small quantities of VG (Dumas et al., 1982); this method, however, is more time-consuming or requires specific equipment. For calculating absolute VG values from A280nminto mg/ml we determined a conversion factor of 1.6. This factor is somewhat higher than that found for vitellin of Locusta mi-

Vitellogenin in Nauphoeta cinerea

71

gratoria (1.1) by measuring the molar extinction with the onset of gestation and remains high for its coefficient (Gellissen et al., 1976). For Leucophaea duration. maderae vitellin Dejmal and Brooks (1972) found The haemolymph protein concentration fluctuates E280nm = 0.83 cm2/mg which corresponds to a con- markedly throughout the oocyte maturation cycle, version factor of 1.2. The differences observed might being high until day 8 and declining until ovulation (Fig. 7). Between day 9 and ovulation the VG and be due to some extent to methodical differences. The haemolymph titer of VG is dependent on the protein titer changes are very similar (Fig. 7), indirates of its synthesis and release from the fat body cating that at this time VG is the major component and its incorporation into the oocytes. Our causing protein content fluctuations. Obviously the measurements of VG and total protein in the concentration changes cannot be due to changes in haemolymph of individual females revealed a high haemolymph volume since we found that the latter variability between insects of the same age and varied very little (Meyer, unpublished). The protein physiological stage (Fig. 7). The haemolymph VG content data (Fig. 7) are similar to those reported for titer and the ovary dry weight are practically not Nauphoeta by Biihlmann (1976) using other techcorrelated (Btihlmann, 1976; Buschor, unpublished), niques. Similar fluctuations to those in Nauphoeta a finding different from that in Schistocerca gregaria have been observed in Leucophaea maderae (Engelmann and Penney, 1966) and Diploptera punctata where the two parameters are correlated (Injeyan and Tobe, 1981). In accordance with Biihlmann (1976), (Mundall et al., 1981). In contrast, in Periplaneta we thus postulate a pulsating VG release and/or americana the prtotein concentration remains at a high level during the entire oocyte maturation period uptake in Nauphoeta. The general course of the haemolymph VG titer and only drops significantly during egg-case forcurve (Fig. 7) is very similar to that obtained for mation (Bell, 1969). The maximum proportion of VG in total protein Nauphoeta by Biihlmann (1976) measuring relative VG concentrations in pools of 5 females with a as observed at ovulation is high in Nauphoeta (58%, radioimmunoassay. The increase in VG concen- Fig. 7) compared to Diploptera punctata with 4.6% tration at the time of chorion formation, when VG is shortly before ovulation (Mundall et al., 1981), while no longer incorporated into the oocytes, indicates during vitellogenesis VG makes up 12% of the total that VG is still released from the fat body. This is protein in Nauphoeta (Fig. 7) and 5-6% in Leucoprobably due to the still high JH titer (Lanzrein et al., phaea maderae (Engelmann, 1978). The methods and data presented here now allow us 1981). A comparison of the VG titers and their fluctuations among different species of cockroaches to study a possible involvement of VG in corpora reveals remarkable differences related to the type of allata regulation by using a newly developed techreproduction (ovoviviparous, oviparous, viviparous). nique for their long-term culture (Wilhelm and LanzIn the ovoviviparous species Byrsotria fumigata the rein, in preparation), as well as the mechanisms and concentration of VG A and VG B shows two peaks, control of VG uptake. a first one at the time when oocytes grow slowly and a second immediately before ovulation. It drops Acknowledgements--We wish to thank Mr P. Beyeler for rapidly and the VG can no longer be detected during technical assistance, Mr M. Kaltenrieder for drawing the pregnancy (Barth and Bell, 1970). In another ovo- graphs and Mrs A. Tschan for rearing the cockroaches. viviparous cockroach, Leucophaea maderae, Brooks Thanks are also due to Dr W. Riesen (Tiefenau Hospital, (1976) and Koeppe and Wellman (1980) showed that Berne) for providing the facilities for lipoproteinelectrophoresis and to Dr G. B/ihlmann (Federal Dairy the fat body in vitro releases VG almost until the Research Station, Berne) for antibodies. Financial support ootheca is formed and Engelmann (1969) reported by the Swiss National Sciences Foundation (grants 3.188.77 the absence of VG in haemolymph during pregnancy. and 3.714.80) is gratefully acknowledged. Obviously the data reported in these ovoviviparous species (Nauphoeta einerea, Byrsotria fumigata and Leucophaea maderae) are very similar. In contrast, in REFERENCES the oviparous species Periplaneta americana the haemolymph VG concentration remains high around Axelsen N. 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