550 A.C.A. Melo et al. Insect Biochemistry and Molecular Biology 30 2000 549–557
Lauverjat et al., 1984 and mosquitoes Raikhel and Lea, 1991. These channels, which are transient, undergo
dramatic changes in patency in Rhodnius during oocyte maturation and this change correlates perfectly with the
rate of vitellogenin uptake Oliveira et al., 1986.
In addition to accumulating Vg synthesized by the fat body the ovaries also synthesize proteins that are
required for embryonic development. The commitment of follicle cells to this process is well known Melius
and Telfer, 1969; Anderson and Telfer 1969, 1970. In some organisms the ovary has been described as the site
of synthesis of non-vitellin yolk proteins such as paravit- ellogenin in Hyalophora cecropia Telfer et al., 1981
and egg-specific protein in Bombyx mori Irie and Yama- shita, 1983. Synthesis of vitellogenin by ovaries so far
has been demonstrated only in higher Diptera and some Coleoptera Postlethwait et al., 1980; Bownes, 1982;
Brennan et al., 1982; Fourney et al., 1982; Zhai et al., 1984; Bianchi et al., 1985; Peferoen and De Loof, 1986;
Zongza and Dimitriadis, 1988. To our knowledge there are no reports bearing on the possibility of Vg synthesis
by ovarian tissues of Hemiptera.
In this work, the ovaries of the hemipteran Rhodnius prolixus are shown to synthesize Vg. Follicle cells sur-
rounding large oocytes are active in this process, and it is shown that follicle cells continue to produce Vt even
after closure of interfollicular channels, i.e. that are no longer engaged in the uptake of yolk precursors from
the hemolymph. A possible role of R. prolixus ovarian Vg is considered.
2. Materials and methods
2.1. Animals Insects were taken from a colony of Rhodnius prolixus
maintained at 28 °
C and 70–80 relative humidity. The experimental insects were adult mated females fed on
rabbit blood at three-week intervals. 2.2. Tissue preparations
Ovaries were dissected out from females fed two days earlier with rabbit blood and the follicles were examined
under a Zeiss stereomicroscope. Oocyte lengths were measured using an ocular micrometer. To obtain follicle
cells free of oocytes, each follicle was opened up, using iridectomy scissors, and the oocyte cytoplasm discarded,
so that the final preparation was a “lawn” of follicle cells attached to a naked oocyte membrane.
2.3.
32
Pi purification Carrier-free
32
Pi purchased from Comissa˜o Nacional de Energia Nuclear Sa˜o Paulo, Brasil was purified by
means of Dowex 1X-10 ion-exchange column de Meis and Masuda, 1974.
2.4. Incorporation of [
35
S]-methionine or
32
Pi into proteins
Isolated ovaries or follicle cells were incubated in Dulbecco’s Modified Eagle’s medium DME-M3916,
Sigma, St Louis containing 0.1–0.2 µ
Ci µ
l [
35
S]-meth- ionine catalog no. 51006 ICN Pharmaceuticals, Inc, Irv-
ine, California or purified
32
Pi 0.15 µ
Ci µ
l in a phos- phate-free culture medium DME-D3656, Sigma, St
Louis at 28 °
C for 90 min. Subsequently, the tissues were set aside for determination of total protein accord-
ing to Lowry et al. 1951 and protease inhibitors 1 mM benzamidine; 0.05 mgml leupeptin; 0.05 mgml SBTI;
0.05 mgml LBTI were added to the medium. The radioactive proteins secreted to the culture medium were
analyzed by SDS-PAGE followed by autoradiography or immunoprecipitated with antiserum raised against Vt. In
some experiments the proteins secreted to the medium were precipitated with 15 TCA wv washed several
times and the radioactivity estimated by scintillation counting. The data were expressed as [
35
S] incorporated per microgram of tissue protein.
2.5. Polyacrylamide gel electrophoresis Electrophoresis was performed in the presence of SDS
Laemmli, 1970 in a 7.5 polyacrylamide gel or using a polyacrylamide gradient 6 to 22, followed by
staining with Coomassie Brilliant Blue. The gels were destained using a mixture of 7 acetic acid and 40
methanol. Molecular masses were estimated using the following protein standards for SDS-PAGE Sigma, St
Louis: myosin 205 kDa,
β -galactosidase 116 kDa,
phosphorylase b 97 kDa, albumin 66 kDa, ovalbumin 45 kDa, glyceraldehyde-3-phosphate dehydrogenase
36 kDa, carbonic anhydrase 29 kDa, trypsinogen 24 kDa, soybean trypsin inhibitor 20 kDa and
α -lactal-
bumin 14 kDa. 2.6. Vitellin purification
Chorionated oocytes were homogenized at 4 °
C in 10 mM Tris–HCl pH 7.0 containing a mixture of protease
inhibitors with concentration of 0.05 mgml of soybean trypsin inhibitor, leupeptin and 1 mM benzamidine and
centrifuged for 5 min at 4 °
C. To the oocytes homogenate supernatant ammonium sulphate was added to final
concentration of 45. The precipitated proteins were removed by centrifugation and discarded. The concen-
tration of the ammoniun sulphate was increased up to 60 and the precipitate containing vitellin was collected
and washed twice in the same solution. The washed pre- cipitate was re-suspended in 45 ammoniun sulphate
551 A.C.A. Melo et al. Insect Biochemistry and Molecular Biology 30 2000 549–557
and dialyzed overnight against 10 mM Tris–HCl pH 7.4 containing 0.15 M NaCl. The proteins were concentrated
on speed-vac concentrator and applied onto a column 90
× 1.5 cm of Sephacryl S-200 HR. The peak of Vt
was collected and re-applied onto the same column but now eluted in a 10 mM Tris–HCl pH 7.4 containing 0.3
M NaCl. The purity of the sample was monitored by SDS-PAGE and only the subunits of Vt were visible.
2.7. Antiserum Purified vitellin 1 mg suspended in complete
Freund’s adjuvant was injected subcutaneously in the back of a 1.5 kg rabbit. Two weeks after injection, a
booster was given; 30 days later blood was taken from an ear vein and the serum examined by Western blotting
Towbin et al., 1979 using an adult female’s hemo- lymph.
2.8. Immunoprecipitation For immunoprecipitation, samples 100
µ l were pre-
incubated with pre-immune serum 20 µ
l during 60 min at 4
° C. Then, 20
µ l of protein A-agarose P7786, Sigma,
St Louis was added and allowed to react for 60 min at 4
° C. The samples were centrifuged at 12,000 g for 5
min. To the supernatant 20 µ
l of specific serum raised against Vt was added, followed by incubation during 180
min at 4 °
C. After addition of 30 µ
l of protein A-agarose, the samples were centrifuged at 12,000 g for 5 min. The
immunoprecipitate was washed three times with 50 mM Tris pH 8.0, 0.5 M NaCl, 1 NP40 octylphenol-
polyethoxyethanol and counted.
2.9. Immunoblotting The proteins were separated by SDS-PAGE 7.5
polyacrylamide, during 180 min at 2 mAcm and then electrotransferred to a nitrocellulose membrane in 25
mM Tris, 192 mM glycine, 20 methanol pH 8.3 for 120 min at 150 mA, followed by staining with Ponceau
Red or preparation for immunostaining as follows: the membrane was incubated with antiserum raised against
purified vitellin followed by secondary antibody conju- gated with alkaline phosphatase and developed with
NBTIBCIP Towbin et al., 1979. After immunostaining the membrane was washed several times with water,
dried at room temperature and autoradiographed using XAR5 film Sigma, St Louis.
2.10. Trichloroacetic acid protein precipitation The radioactive samples culture medium containing
radioactive proteins secreted by follicle cells were placed on filter paper and dried at room temperature. The
filter papers were washed once with cold 15 trichlo- roacetic acid for 15 min, twice with cold 10 tricholo-
roacetic acid and finally with 70 ethanol Sahal and Yamaguchi-Fujita, 1987. The papers were dried and the
radioactivity estimated by scintillation counting.
2.11. Analysis of radioactive vitellogenin by ion- exchange chromatography
Culture medium obtained from follicle cells incubated with [
35
S]-methionine or
32
Pi was dialyzed during 8 h at 4
° C against buffer A 20 mM Tris–HCl pH 8.4, 2 mM
EDTA, 2 mM EGTA, 5 mM NaN
3
to remove [
35
S]- methionine or
32
Pi not associated with proteins. The dialysate was then applied onto a DEAE-Toyopearl 650
M column 1.4 ×
24 cm equilibrated in buffer A. The column was washed with buffer A, a linear gradient from
0.1 M to 0.2 M NaCl in buffer A was developed and the fractions collected. Each fraction was examined in a
spectrophotometer at 280 nm and an aliquot counted in a scintillation counter.
2.12. Immunofluorescence Ovarioles were isolated and fixed using 4 paraform-
aldehyde in PBS. The fixed preparations were allowed to adhere to cover glasses coated with poly-l-lysine, then
washed with PBS and treated with 150 mM NH
4
Cl dur- ing 20 min. Permeation was obtained by treatment with
0.1 Triton X-100 in PBS, for 5 min at room tempera- ture. Non-specific staining was avoided by treatment
with PBS containing 1.5 albumin and 0.5 gelatin blocking buffer during 30 min. After incubation with
antiserum raised against vitellin diluted 1:500 for 60 min, preparations were washed with blocking buffer and
finally incubated with goat anti-rabbit secondary anti- body associated with fluorescein Gibco, Grand Island,
N.Y diluted 1:100 in blocking buffer, for 60 min in the dark. The preparation was mounted with 0.2 M n-propyl
gallate in 9:1 glycerol-PBS and analyzed using a Zeiss laser scanning microscope LSM 310, operating in non-
confocal mode. Obtained images were all equally pro- cessed using Adobe Photoshop.
2.13. Immuno-electron microscopy localization For electron microscopy analysis vitellogenic follicles
were fixed in a mixture of 0.1 glutaraldehyde type I and 4 paraformaldehyde in PBS pH 7.2 for 2 h at
room temperature. After fixation, oocytes were washed in PBS and then incubated with 50 mM glycine in PBS
for 60 min, washed in PBS and dehydrated in a series of methanol solutions 30 to 90 and finally embed-
ded in Unicryl British Biocell at 220
° C under UV
illumination. Ultrathin sections were collected on 300 mesh nickel grids. The sections were subsequently incu-
bated in PBS pH 7.4 containing 150 mM NH
4
Cl for
552 A.C.A. Melo et al. Insect Biochemistry and Molecular Biology 30 2000 549–557
30 min, PBS containing 1.5 albumin, 0.5 of gelatin and 0.1 Tween 20 blocking buffer for 30 min, and
primary antibody raised against vitellin for 60 min diluted 1:500. Afterwards, sections were washed in
blocking buffers, incubated with 10 nm gold-labeled goat anti-rabbit IgG 1:100 Sigma Chemical Co for
60 min, and thoroughly washed in PBS. Grids were examined in a Zeiss 900 electron microscope, after stain-
ing with uranyl acetate and lead citrate. Control experi- ments demonstrating specificity were performed using
non-immune serum followed by incubation with gold- labeled goat-anti-rabbit IgG.
3. Results
