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
3.1. Follicle cells as a site of synthesis of vitellogenin When Rhodnius prolixus ovaries, with their associated
follicles, were incubated in a culture medium containing [
35
S]-methionine at 28 °
C, radioactive vitellogenin and other proteins were synthesized and secreted to the
medium Fig. 1. In order to localize the site of vitellog- enin synthesis in the ovary, follicular epithelium cells
free of oocytes were incubated in the same culture medium. Analysis of the secreted proteins by SDS-
PAGE and Western blots shows that the main protein secreted to the culture medium by the whole ovary and
by the follicle cells is radioactive Fig. 1C and corre- sponds to vitellogenin, since it is recognized by anti-
serum raised against vitellin Fig. 1A and B. This experiment demonstrates that follicle cells are a site for
synthesis of this yolk protein.
Rhodnius vitellogenin and vitellin are known to be
Fig. 1. Analysis of vitellogenin synthesized by the ovary and follicular epithelium cells of Rhodnius prolixus. A SDS-PAGE stained by Coomassie
Blue R. B Western blot. C Autoradiography of the nitrocellulose membrane in B. Follicle cells or ovaries were incubated for 90 min in DME culture medium in the presence of [
35
S]-methionine. The proteins secreted to in the culture medium were separated by SDS-PAGE A, 7.5 acrylamide, transferred to a nitrocellulose membrane, and challenged with antibody raised against vitellin and developed using a secondary antibody
conjugated with phosphatase B, followed by autoradiography C. Molecular weights of the four vitellin subunits are indicated at the left. Lane 1: non-radioactive extract of chorionated oocyte; lane 2: proteins synthesized and secreted to the culture medium by a follicle-cell preparations
isolated from around 20 oocytes; lane 3: proteins synthesized and secreted to the medium by three intact ovaries. Fig. 2.
Purification of ovarian vitellogenin secreted by the follicle cells. The follicle cells were incubated with [
35
S]-methionine I or
32
Pi s for 90 min and the proteins secreted to the culture medium separated on a DEAE-Toyopearl column see Materials and Methods.
The protein content was estimated from the absorbance at 280 nm —. The radioactivity associated with proteins was estimated by scintil-
lation counting. Inset Autoradiography of SDS-PAGE of [
35
S] pro- teins synthesized and secreted by follicle cells: Lane 1: total protein,
lane 2: protein immunoprecipitated with antiserum against vitellin, prior to application on the column.
553 A.C.A. Melo et al. Insect Biochemistry and Molecular Biology 30 2000 549–557
phosphorylated lipoglycoproteins Masuda and Oliveira, 1985. To ascertain whether the vitellogenin synthesized
by follicle cells is also phosphorylated, parallel experi- ments were conducted with cells incubated in culture
medium enriched with
32
Pi or [
35
S]-methionine followed by separation of the secreted proteins on a DEAE-Toy-
opearl column Fig. 2. The experiment shows that a major radioactive peak is labeled with both [
32
P] and [
35
S]. Analysis by SDS-PAGE of total radioactive pro- teins or immunoprecipitated with antiserum against vitel-
lin also shows that the main protein in fact correspond to vitellogenin inset.
3.2. Localizing the cells responsible for synthesis In order to determine whether vitellogenin synthesis
occurs in specialized cells of the follicular epithelium, antiserum against vitellin was raised in rabbits and used
Fig. 3. Immunofluorescence of a follicle challenged with serum raised against vitellin. The follicles 1.5 mm to 1.7 mm in length was fixed with
4 paraformaldehyde in PBS, followed by washing with 150 mM NH
4
Cl and then 1.5 albumin plus 0.5 of gelatin wv. The preparation was challenged with serum raised against vitellin and then with goat anti-rabbit secondary antibody associated with fluorescein and visualized in a
confocal laser scanning microscope conventional fluorescence mode. Follicle cells and oocyte are indicated on the figure. A Phase-contrast micrograph; B Control of immunofluorescence of similar follicle treated with secondary antibody; C immunofluorescence of follicle treated
with antiserum raised against vitellin; D Superimposition of images A and C in a RGB system. Red channel corresponds to immunofluorescence presence of Vg and Vt. Green channel corresponds to phase-contrast micrograph.
for immunolocalization. Goat anti-rabbit antibody lab- eled with fluorescein Fig. 3 or gold particles Fig. 4
were used. Independent of the technique used, vitellog- enin was found inside the great majority of follicle cells
analyzed and also, as expected, inside the oocyte. Except for the follicle cells found close to the area between two
oocytes, where a fluorescent signal of different intensity was observed, no other specialization could be detected
among the other follicle cells Fig. 3, Fig. 4. Controls, using secondary antibody, were performed for both tech-
niques. No reactions was observed either by immuno- fluorescence Fig. 3 or immunogold detection data
not shown.
To further analyze the capacity of the follicle cells to synthesize proteins, epithelial cells obtained from fol-
licles of different sizes were incubated in the culture medium, and the total radioactivity associated with
secreted proteins was estimated after acid precipitation
554 A.C.A. Melo et al. Insect Biochemistry and Molecular Biology 30 2000 549–557
Fig. 4. Immunocytochemical localization by transmission electron microscopy of vitellogenin in sectioned follicles embedded in Unicryl. Anti-
vitellin immune serum was used as primary antibody, followed by incubation with 10 nm gold-labeled goat anti-rabbit IgG. Arrow indicate representative gold particles. The follicle with associated oocyte 1.5 to 1.7 mm in length was selected from ovary dissected two days after a
blood meal. A A view inside the follicle cell FC ×
60,000. B Detail of O-Vg secretion between two follicle cells ×
112,500. indicates intercellular space. C Panoramic view of the region between a follicle cell FC and oocyte O
× 21,000. D A view inside the oocyte showing
yolk granule Y and cytoplasm cy ×
55,000. FC Follicle cell; MV Microvilli; O Vitellogenic oocyte.
Fig. 5. The figure shows that the epithelial cells of small follicles are much more active with respect to total
protein synthesis than cells of follicles of larger size. In order to determine when the synthesis of vitellogenin
occurs, the proteins secreted by epithelial cells derived from follicles of different sizes were analyzed by SDS-
PAGE, and autoradiographed Fig. 6A. The experiment shows that vitellogenin synthesis occurs primarily in the
larger follicles, suggesting its participation in the late phase of vitellogenesis. Only the subunit of high molecu-
lar mass is visible, probably due to the small amount of methionine in the smaller subunits see Fig. 1B and C.
Additionally the fact that only the largest subunit is vis- ible in Fig. 6 can also be attributed to the quenching of
radioactivity. The autoradiography was obtained expos- ing the film on to dried gel for 30 days while in Fig. 1
where the proteins were previously transferred to nitro- celulose 11 days were enough to sensitize the film and
display the small subunits Fig. 1C. The immunofluo- rescence experiment shown in Fig. 6B and C where
epithelial cells derived from large follicles reacted strongly when challenged with antibody raised against
Vt, but not the epithelial cells derived from small size oocytes is in agreement with the biochemical obser-
vation Fig. 6A.
4. Discussion
