Archives of Insect Biochemistry and Physiology 39:133–143 (1998)

© 1998 Wiley-Liss, Inc.

Uptake of Rhodnius Heme-Binding Protein (RHBP) by theOvary Of Rhodnius prolixus

Ednildo A. Machado,1 Pedro L. Oliveira,1 Monica F. Moreira,1 Wanderley de Souza,2 and Hatisaburo Masuda1*

1Departamento de Bioquímica Médica, Instituto de Ciências Biomédicas, Rio de Janeiro, Brasil2Laboratório de Ultraestrutura Celular Hertha Meyer, Instituto de Biofísica Carlos Chagas Filho,

Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brasil

The uptake of RHBP (Rhodnius heme-binding protein) by theovaries of Rhodnius prolixus was characterized. RHBP puri-fied from oocyte was labeled with 125I and used to study theprocess of uptake by the ovary in vivo and in vitro. After injec-tion, the [125I]RHBP was readily removed from the hemolymphand accumulated especially in the ovary. The capacity of theovary to take up [125I]RHBP from the hemolymph varied dur-ing the days following blood meal. It increased up to day 2,remained stable until day 5, and then decreased up to the endof oogenesis. In vitro, the uptake of [125I]RHBP was linear atleast up to 60 min. The uptake was dependent on [125I]RHBPconcentration and showed to be a saturable process. The addi-tion of a molar excess of non-related proteins such as Vitellin(Vt), Lipophorin (Lp), and Bovine Serum Albumin (BSA) didnot reduce [125I]RHBP uptake. Using immunogold technique theRHBP was localized at the microvilli, coated pits, and yolk gran-ules. The main yolk protein, Vt, did not compete with RHBPfor the uptake. Thus, it is discussed here that they bind to in-dependent binding sites of the oocytes, and are directed lateron to the same compartment. The need of both proteins for thecompletion of mature oocyte was verified in vivo. The reduc-tion of heme-RHBP in the hemolymph, by changing the diet,decreased the number of eggs laid. Increasing the concentra-tion of heme-RHBP in the hemolymph, the number of eggs pro-duced increased in a dose dependent manner. In vitro, bothapo-RHBP and heme-RHBP can be taken up by the oocyte.Since the mature oocyte contains only heme-saturated RHBP,the possible fate of apo-RHBP is also discussed. Arch. InsectBiochem. Physiol. 39:133–143, 1998. © 1998 Wiley-Liss, Inc.

*Abbreviations used: apo-RHBP = RHBP free of heme; BSA= Bovine Serum Albumin; heme-RHBP = RHBP associatedwith heme; [125I]RHBP = 125I – labeled RHBP; Lp = lipop-horin; Mvg = microvitellogenin; PBS = phosphate bufferedsaline; RCBP = Rhodnius calcium-binding protein; rRHBP= reconstituted heme-RHBP; RHBP = Rhodnius heme-bind-ing protein; Vg = vitellogenin; Vt = vitellin.

Contract grant sponsor: Conselho Nacional de Desen-volvimento Científico e Tecnológico (CNPq); Contract grantsponsor: Financiadora de Estudos e Projetos (FINEP); Con-

tract grant sponsor: Programa De Apoio ao DesenvolvimentoCientífico e Tecnológico (PADCT); Contract grant sponsor:Programa de Núcleos de Excelência (PRONEX).

*Correspondence to: Hatisaburo Masuda, Universidade Fed-eral do Rio de Janeiro, Instituto de Ciências Biomédicas,Departamento de Bioquímica Médica, Cidade Universitária- Ilha do Fundão, 21.944-590, Rio de Janeiro, RJ, Brasil. E-mail: masuda@server.bioqmed.ufrj.br

Received 4 May 1998; accepted 25 September 1998

134 Machado et al.

Key words: oogenesis; endocytosis; Rhodnius prolixus; RHBP; heme

INTRODUCTION

Vitellogenesis is characterized by massiveaccumulation of yolk proteins by the insect ova-ries. The best-studied yolk protein is vitellogenin(Vg*), a high molecular mass lipoglycoprotein syn-thesized in the fat body. It is taken up from thehemolymph by the growing oocytes through re-ceptor-mediated endocytosis (Telfer, 1961; Rothand Porter, 1964; Engelmann, 1979; Raikhel andDhadialla, 1992; Valle, 1993). The formation of aspecialized cortex, the endocytotic complex, thatincludes microvilli, coated pits, coated vesicles,and endosomes renders the oocyte competent forprotein uptake (Roth and Porter, 1964; Huebnerand Anderson, 1972; Raikhel and Dhadialla,1992). Once inside the oocytes, Vg is then referredas vitellin (Vt) (Engelmann, 1979). In the last fewyears, several non-Vt yolk proteins such asmicrovitellogenin (Mvg) (Kawooya et al., 1986),lipophorin (Lp) (Kawooya et al., 1988; Van An-twerpen et al., 1993), carboxypeptidases (Cho etal., 1991), insecticyanin (Kang et al., 1995), andRhodnius calcium binding protein (RCBP) (Silva-Neto et al., 1996) have been described. The en-docytotic pathways of these proteins are stillpoorly characterized as compared with Vt.

In Rhodnius prolixus, Oliveira et al. (1995)described a 15-kDa protein found in hemolymphand oocytes and named it RHBP (Rhodnius heme-binding protein). In the hemolymph, RHBP is notsaturated with heme and thus is promptly ableto bind heme. As a consequence, RHBP functionsas an antioxidant agent, inhibiting heme-inducedlipid peroxidation (Petretski et al., 1995). Thus,in the hemolymph RHBP can be found both inthe form of a hemeprotein, with iron-protoporphy-rin IX bound (heme-RHBP), and as the apopro-tein, without heme (apo-RHBP). During digestionof blood, heme is released from hemoglobin,crosses the digestive barrier and reaches thehemolymph, where it is sequestered by apo-RHBPconverting it to heme-RHBP (Petretski et al.,1995). In contrast, RHBP isolated from oocytesis totally composed of heme-RHBP, and is not ableto bind additional heme (Oliveira et al., 1995).The role of RHBP in the oocytes is still unknown.

In the present article we report the uptakeof RHBP by vitellogenic ovaries of the blood-suck-ing bug Rhodnius prolixus. We present evidencethat RHBP is incorporated into growing oocytesby endocytosis mediated by a specific receptor,

and also that it ends up in yolk granules, thesame compartment where Vt is stored. The routeof entry of these proteins and the need of bothfor the final composition of the oocyte are alsodiscussed.

MATERIALS AND METHODS

Insects

Insects were taken from a colony of Rhodniusprolixus maintained at 28°C and 70–80% relativehumidity. The insects used were normal matedfemales having their second blood meal as anadult. Experimental animals were fed with rab-bit blood using an artificial feeding apparatus(Garcia et al., 1975).

Protein Purification

Lipophorin (Lp). Lipophorin was purified ac-cording to the procedure described by Gondim etal. (1989a).

Vitellin. For Vt isolation, chorionated oocyteswere homogenized in NaCl 0.15M, centrifuged at11,000g for 5 min. The pellet and the floating lip-ids were discarded. The oocyte homogenate wasdialyzed overnight at 4°C against buffer A (1 mMEDTA, 1 mM EGTA, 0.02% NaN3, 20 mM Tris-HCl, pH 8.0), and applied to a DEAE Toyopearl650 M column (1.4 X 24 cm) equilibrated in thesame buffer. The column was eluted with 50 mlof linear gradient of NaCl (0.2–0.4 M) in bufferA. The fractions containing Vt were collected andpooled for further analysis.

Oocyte RHBP. Oocyte RHBP was purifiedfrom oocytes according to Oliveira et al. (1995).The procedure was modified as follows: The oo-cytes were homogenized and centrifuged as de-scribed for Vt isolation. The oocyte homogenatewas dialyzed overnight at 4°C against buffer A (1mM EDTA, 1 mM EGTA, 0.02% NaN3, 20 mMTris-HCl, pH 8.0), and applied to a DEAE Toyo-pearl 650 M column (1.4 X 24 cm) equilibrated inthe same buffer. The column was eluted with 50ml of linear gradient of NaCl (0–0.1M) in bufferA. The RHBP containing fractions were pooled,and concentrated in a Speed-Vac System (SavantInstruments, Inc., Farmingdale, NY). The degreeof purification of Vt, Lp and RHBP was moni-tored by SDS-PAGE using the basic procedure ofLaemmli (1970) and O’Farrell (1975) buffers and

Uptake of RHBP by the Ovary 135

solutions. Protein concentration was estimatedaccording to Lowry et al. (1951) using BSA (bo-vine serum albumin) as standard.

Labeling RHBP With 125I

Oocyte RHBP was radiolabeled by additionof 125I (10–100 µCi) to an Iodo-Gen-coated tubewith 100–700 µg of purified protein in 0.5 ml ofwater plus 0.5 ml of phosphate-buffered saline(10 mM phosphate and 0.14M sodium chloride).After 20 min at 4°C, the reaction was stopped byextensive dialysis against water at 4°C. The iodi-nation was performed according of the Iodo-Genmethod (Watt et al., 1979). The specific activityof [125I]RHBP (125I -labeled RHBP) was estimatedby γ counting.

Uptake of [ 125I]RHBP by the Ovary In Vivo

Purified [125I]RHBP (1–5 µg protein; 100,000–200,000 cpm in 1–5 µl 0.15M NaCl) was injectedwith a 5 µl Hamilton (Reno, NV) syringe into thethorax of vitellogenic females. Unless otherwisestated, after injection the experimental animals weremaintained at 28°C until dissection. At indicatedtimes, ovaries and other organs were dissected, ex-tensively washed in 0.15M NaCl, and the radioac-tivity incorporated by different organs was estimatedby γ counting after homogenization in 0.15M NaCl(Gondim et al., 1989b; Machado et al., 1996).

Uptake of [ 125I]RHBP by Isolated Ovaries

On the second or third day after the bloodmeal, the ovaries were dissected, washed in 0.15MNaCl, and incubated at 28°C or 0°C in culturemedium (Eagle’s, Sigma Chemical Co, St. Louis,MO) containing purified [125I]RHBP (2 ovaries 50µl of medium). After the incubation period, ova-ries were left for an extra 30 min in 1 ml ofRhodnius physiological solution (Maddrell, 1969)at 28°C. At the end of this post-incubation thetracheae adhering to the ovary, the ovarian sheathand the oviducts were carefully removed. The ra-dioactivity incorporated by each ovary was deter-mined by γ counting after homogenization in0.15M NaCl.

Preparation of Radioactive apo-RHBP

Radioactive apo-RHBP was prepared by theextraction of heme from purified RHBP previouslylabeled with 125I (Iodo-Gen method) using acetone-HCl (Schichi and Hacket, 1962). The rRHBP(reconstituted heme-RHBP) was obtained by a con-tinuous addition of hemin to the [125I]apo-RHBP, upto the point of saturation, in 0.01M phosphate

buffer, pH 7.0, containing 0.15 M NaCl (PBS). Theuptake of radiolabeled apo-RHBP and rRHBP invitro was performed as described for RHBP.

Antiserum

Purified RHBP (0.5 mg) or Vt (0.5 mg) emul-sified in complete Freund’s adjuvant was injectedsubcutaneously in the back of a 2-kg rabbit. Abooster was given 3 weeks after the first injec-tion. After 1 month, the blood was taken from anear vein and the serum specificity was checkedby Western blot (Towbin et al., 1979). IgG wasfurther purified by an immunoaffinity protocoldescribed earlier (Oliveira et al., 1989).

Immunocytochemical Localization

Vitellogenic follicles were fixed in 1% glut-araldehyde (Sigma type I), 4% paraformaldehyde,1 mM CaCl2, 0.1 % picric acid, 3.5% sucrose in0.1 M cacodylate buffer, pH 7.2. After fixation,the oocytes were washed in 0.1 M cacodylatebuffer, pH 7.2, 3.5% sucrose, and 1 mM CaCl2,followed by incubation in the same buffer con-taining 50 mM NH4Cl for 60 min at room tem-perature for aldehyde group blocking. Afterwashing in 0.1 M cacodylate buffer, pH 7.2, 3.5%sucrose, and 1 mM CaCl2, the material was incu-bated for 2 h in 2% uranyl acetate in 15% ac-etone. Finally, the material was dehydrated inacetone, and embedded in the hydrophilic resinLR White (Polysciences, Inc. Warrynton, PA) ac-cording to Berryman and Richard (1990). Thinsections were made and collected on 300 meshnickel grids. Grids were incubated for 1 h in (adrop) 0.01 M phosphate-buffered saline (PBS), pH7.4, containing 5% non-fat milk. They were thenincubated in a drop of anti-RHBP or anti-Vt an-tibodies diluted (1:100) in PBS containing non-fat milk for 60 min at room temperature. Afterwashing in PBS containing non-fat milk, theywere incubated with gold-labeled Goat anti-rab-bit IgG (1:500), obtained from Sigma ChemicalCo., for 1 h at room temperature. They were thenthoroughly washed in PBS, rinsed in distilled wa-ter, and dried. In order to examine the samplesin a Zeiss (Thornwood, NY) 900 electron micro-scope, the sections were stained with uranyl ac-etate and lead citrate. Control experimentsdemonstrating specificity were performed usingnon-immune serum followed by incubation withgold-labeled Goat-anti rabbit IgG (Machado et al.,1996). The mild fixation of the oocytes preservesthe antigenicity, although ultrastructural detailsof internalization route could not be discerned.

136 Machado et al.

Effect of Different Levels of Heme-RHBP onOocyte Maturation

To investigate the effect of different levelsof heme-RHBP on the number of eggs produced,the insects were fed with the same volume of rab-bit blood, plasma, and plasma enriched with 1mM hemin in an artificial feeding apparatus(Garcia et al., 1975). (During 16 days after thismeal, the eggs were collected, and counted dailyfor 16 days). To analyze the effect of differentmeals offered to the insects on the profile of pro-teins, the hemolymph of five females of eachgroup was collected, monitored by SDS-PAGE us-ing the basic procedure of Laemmli (1970) andusing O’Farrell (1975) buffers and solutions. Ashas been described, diets containing different con-

centrations of hemin alter the level of heme-RHBP in the hemolymph (Petretski et al., 1995).

RESULTS

Uptake of [ 125I]RHBP by the Ovary In Vivo

After injection into vitellogenic females,[125I]RHBP was readily removed from hemolymphand accumulated in the ovary (Fig. 1). When thefemales were kept at 4°C after [125I]RHBP injec-tion, the uptake was reduced, suggesting the de-pendence on active metabolism (data not shown)similar to that reported for other endocytotic pro-cesses (Raikhel and Dhadialla, 1992). The ovarywas the major site of [125I]RHBP accumulation(Fig. 2). The capacity of the ovary to take up

Fig. 1. Kinetics of radioactivity disappearance from thehemolymph and simultaneous appearance in the ovaries, fol-lowing injection of purified [125I]RHBP. At different timesafter injection into vitellogenic females maintained at 28°C,1 µl of hemolymph (4) was collected and the radioactivityestimated in a γ counter. Simultaneously the ovaries (2) wereremoved, washed, and homogenized and the radioactivityestimated in a γ counter. Data are mean ± S.E for five de-terminations.

Fig. 2. Distribution of the radioactivity from [125I]RHBPamong different organs in vivo. Four hours after injectionof purified [125I]RHBP into vitellogenic females maintainedat 28°C, the different organs indicated (OV: ovary; H: heart;AM: anterior midgut; PM :posterior midgut; SG: salivarygland; FB: fat body; MT: malphigian tubules) were dissected,washed, and homogenized in 0.15M NaCl. The radioactivitypresent in each organ was estimated in a γ counter. Otherexperimental conditions were as described in Materials andMethods Data are mean ± S.E for five determinations.

Uptake of RHBP by the Ovary 137

[125I]RHBP was very high at day 2 after bloodmeal, remained stable until day 5, and then de-creased (Fig. 3).

Uptake of [ 125I]RHBP by Isolated Ovaries

The time-course of [125I]RHBP uptake in vitrois shown in Figure 4. At 28°C the [125I]RHBP wastaken up by the ovary at a constant rate, for atleast 1 h. The uptake of [125I]RHBP was depen-dent on its concentration in the medium and satu-rated around 3 mg/ml (Fig. 5), which suggests thepresence of a receptor. The uptake was impairedat low temperatures (Table 1), and the additionof non-radioactive oocyte RHBP to the incubationmedium reduced both uptake (measured at 28°C)and/or binding (measured at 4°C) to the ovaries.The addition of a molar excess of other non-re-

lated proteins as Vg, Lp, and BSA had a small orno effect (Table 1). These results suggested theexistence of a specific receptor for RHBP.

Uptake of [ 125I]apo-RHBP by Isolated Ovaries

Due the presence of apo-RHBP in the hemo-lymph, the uptake of this molecule was studiedin vitro. Table 2 shows that both apo-RHBP andheme-RHBP can be taken up by the oocyte, andare dependent on temperature. Reconstitutedheme-RHBP is also taken up by the oocyte, sug-gesting that the treatment did not influence onits recognition.

Localizing RHBP in the Oocytes

Immunolocalization with specific antibod-ies showed that RHBP was found at the oocyte

Fig. 3. Uptake of [125I]RHBP in vivo by the ovaries on dif-ferent days after feeding. Four hours after injection of puri-fied [125I]RHBP into vitellogenic females, maintained at 28°C,the ovaries were dissected, washed, homogenized, and ra-dioactivity present estimated in a γ counter. Other experi-mental conditions were as described in Materials andMethods. Data are mean ± S.E for five determinations.

Fig. 4. Time course of [125I]RHBP uptake in vitro. Ovarieswere incubated in the presence of [125I]RHBP (1.8 mg/ml) at28°C. At different times, the ovaries were removed, washed,homogenized, and the radioactivity determined in a γcounter. Other experimental conditions were as describedin Materials and Methods. Data are mean ± S.E for fivedeterminations.

138 Machado et al.

TABLE 2. Uptake of [125I]apo-RHBP andReconstituted [125I]heme-RHBP by the Ovary*

Uptake BindingProtein (pmol/ovary) (28°C) (pmol/ovary) (4°C)

Apo-RHBP 15.3 ± 0.46 2.3 ± 0.2rRHBP 17.6 ± 0.86 1.93 ± 0.46Untreated RHBP 14.2 ± 3.6 ND

*Incubations were carried out at 28°C or 4°C for 2 h in cul-ture medium. The ovaries were incubated in the presence of1 mg/ml of untreated [125I]RHBP, [125I]apo-RHBP, and[125I]rRHBP (reconstituted) as indicated. Other experimen-tal conditions were as described in Materials and Methods.The values represent the mean ± standard error of 4 deter-minations. ND = not determined.

TABLE 1. Specific Binding of [125I]RHBP tothe Ovary†

[125I]RHBP uptake [125I]RHBP bindingAddition (28°C) (4°C)

None (control) 0.306 ± 0.02 0.081 ± 0.008Non-radioactive 0.105 ± 0.01* 0.030 ± .006*RHBP (5 mg/ml)BSA (5 mg/ml) 0.278 ± 0.02 NDLp (10 mg/ml) 0.297 ± 0.01 NDVt (4 mg/ml) 0.303 ± 0.01 ND†Incubations were carried out at 28°C or 4°C for 2 h in theculture medium. The ovaries were incubated in the pres-ence of [125]RHBP (0.1 mg/ml) and other proteins as indi-cated. Other experimental conditions were as described inMaterials and Methods. The values represent the mean ±standard error of 4 determinations. ND = not determined.*Significantly different from controls (P < 0.05).

Figs. 6–9. Immunocytochemical localization by transmis-sion electron microscopy of RHBP and Vt. Specific antibodyagainst RHBP (Figs. 6–8) or Vt (Fig. 9) was used as pri-mary antibody followed by gold-labeled goat anti-rabbit IgG.Vitellogenic oocyte (O), microvilli (MV), coated pits (CP), yolkgranules (Y), lipid droplet (LD). Fig. 6, X42,000; Fig. 7,X54,200; Fig. 8, X32,000, and Fig. 9, X18,000. The vitelogenicoocytes were selected from ovaries dissected 2 days afterblood meal.

Fig. 5. Effect of [125I]RHBP concentration on the uptakeby the ovaries. Ovaries were incubated at 28°C in the pres-ence of different concentrations of purified [125I]RHBP(2.600.000 cpm/ nmol of protein). After 40 min, the ovarieswere washed, homogenized, and the radioactivity determinedin a γ counter. The initial points represent 0.1–0.5 mg/ml of[125I]RHBP concentration. Other experimental conditionswere as described in Materials and Methods. Data are ±S.E for four determinations.

surface (microvilli), coated pits (Figs. 6,7) andcoated vesicles (data not shown). Some label-ing was also found inside the oocyte near theplasma membrane. We also observed strong la-beling inside yolk granules (Fig. 8), where Vtis stored (Fig. 9). Besides that, a sequence ofyolk granules fusion can also be seen in Figure8. Controls with non-immune serum indicateda lack of non-specific labeling (data not shown).

Need of Heme-RHBP on Oocyte Maturation

The participation of heme-RHBP and Vt inthe yolk granule formation led us to study apossible relationship of heme-RHBP and Vt onegg production. Figure 10 shows that insectsfed on blood produced five times more eggs thaninsects fed on plasma. Insects fed on plasmaenriched with hemin doubled the number ofeggs produced, when compared with insects fedon plasma alone. SDS-PAGE analysis of hemo-lymph from these insects showed similar con-centrations of all proteins, including Vg andRHBP (Fig 11). Previous work indicated thatunder these conditions we can observe differ-ences on heme-RHBP, while the total amountof RHBP (heme-RHBP + apo-RHBP) remainsconstant (Petretski et al., 1995).

Uptake of RHBP by the Ovary 139

Figures 6–9.

140 Machado et al.

DISCUSSION

As in many other insect species, in Rhodniusprolixus vitellin (Vt) is the major oocyte proteinthat corresponds to 80% of total proteins (Masudaand Oliveira, 1985; Oliveira et al., 1986). In thecase of Rhodnius besides Vt, several non-Vt pro-teins have been described, such as acid hydro-lases (Nussenzveig et al., 1992), RCBP (Silva-Netoet al., 1996), and RHBP (Oliveira et al., 1995).RHBP is the second most abundant protein inRhodnius oocytes, and corresponds approximatelyto half of the amount of Vt in a molar basis (datanot shown). Here we show that [125I]RHBP is ac-cumulated in vitellogenic oocytes by means of re-ceptor-mediated endocytosis (Table 1 and Fig. 5).Several organs and tissues were able to incorpo-rate [125I]RHBP, but the ovary is by far the most

active site for RHBP accumulation (Figs. 1,2). Invitro, the uptake of RHBP by the ovaries was lin-ear at least up to 60 min (Fig. 4) and is a satu-rable process (Fig. 5).

The ovarian capacity to take up [125I]RHBPincreased after blood-meal, reaching a maximumat day 2 (Fig. 3). This is also the period of mostintense uptake of Vg by the growing oocytes inRhodnius prolixus (Oliveira et al., 1986). These ob-servations suggested that the uptake of RHBP andVg was concomitant, and this could be explainedby a common receptor being used for Vg and RHBP.The Vg receptors of Drosophila (Schonbaum et al.,1995) and Aedes aegypti (Sappington et al., 1996)were included in the low-density lipoprotein recep-tor supergene family, which includes low densitylipoprotein receptor-related protein (LRP) (Zdanov-sky et al., 1996), 600 kDa receptor in C. elegansand 100 kDa very low-density lipoprotein receptorin mammals (Schneider, 1996). These receptors aremultifunctional and characteristically bind differ-ent ligands providing to the female germ cell asimple and economical machinery to be used forvitellogenesis. If we extend this concept to the

Fig. 10. Influence of hemin on oviposition. Adult femaleswere fed with blood, plasma with or without 1 mM hemin.During 16 days, the eggs laid by three groups were collecteddaily, pooled, and counted. The cumulative numbers of eggsare represented in the ordinate, and correspond to the aver-age of eggs produced per female. Other experimental condi-tions were as described in Materials and Methods.

Fig. 11. SDS-PAGE of hemolymph proteins after feeding withdifferent meals. Two days after meal, the hemolymph of fe-males fed with blood (lane 1), plasma (lane 2), and plasmaplus 1 mM hemin (lane 3) were collected and pooled. Fromeach sample 1 µl was applied into the gel. The numbers repre-senting the molecular weight were plotted according to theposition of proteins of known molecular weights present in thekit from Sigma. The position of RHBP and sub-units of Vg(vitellogenin) are indicated. Other experimental conditions wereas described in Materials and Methods.

Uptake of RHBP by the Ovary 141

Rhodnius vitellogenin receptor, the lack of com-petition between RHBP and vitellogenin can beinterpreted as the binding of ligands to sepa-rate sites (Table 1); if not we must interpretour results assuming the presence of indepen-dent receptors. The immunolocalization datashowed also the presence of RHBP associatedwith microvilli, coated pits (Figs. 6,7) and coatedvesicles (data not shown), thus characterizing atypical receptor-mediated yolk endocytosis path-way. Besides, RHBP was also found inside inthe yolk granules (Figs. 8,9), which are the sitesof Vt storage (Roth and Porter, 1964; Huebnerand Anderson, 1972). Taken together, our re-sults indicate that although independent bind-ing sites or receptors are used for RHBP andVg uptake, a similar pathway is shared for in-ternalization and storage for both proteins.These data are similar to what happens withother non-Vg yolk proteins as Mvg and Lp ofManduca sexta (Van Antwerpen et al., 1993).

In the frog Xenopus laevis oocyte, the pres-ence of Vg in culture medium has an essentialrole at the targeting and/or fusion events on itsincorporation (Wall and Patel, 1987). In Bombyxmori, an ovary implanted in male hosts producedviable eggs lacking Vg, but presented other non-Vg yolk proteins (Yamashita and Irie, 1980). How-ever, if Vg is not essential in Bombyx, there areno reports addressing the reverse question, i.e.,if non-Vg yolk proteins are obligatory componentsfor correct vitellogenesis and/or embryogenesis.In Rhodnius, females fed on plasma had reducedheme-RHBP levels in hemolymph (Petretski et al.,1995), and reduced number of eggs laid (Fig. 10).The eggs produced by females fed on plasma onlycontained the normal amount of heme-RHBP, andgave rise to normal insects (data not shown).Supplementing plasma with heme resulted in asignificant increase in the number of eggs pro-duced (Fig. 10). Considering that the addition ofhemin to the plasma did not alter the concentra-tions of hemolymph proteins, especially Vg (Fig.11), but altered the heme-RHBP concentrations(Petretski et al., 1995), it is tempting to specu-late that heme-RHBP was a limiting factor forthe production of eggs. In fact, the addition ofhemin to the plasma resulted in an increase inthe number of eggs produced (Fig. 10), in a dosedependent manner (data not shown), indicatingthat the capacity to synthesize Vg was not thelimiting step. Females fed on plasma only wereable to produce around 4 eggs, but doubled thenumber after addition of hemin to plasma. In vitro

the oocytes can also recognize and take up apo-RHBP (Table 2). The conclusion is that the associa-tion of the heme with apo-RHBP is not necessaryfor the uptake, but it is necessary to induce thematuration of the oocyte. If we consider that themature oocyte contain only RHBP saturated withheme (Oliveira et. al. 1995), and the addition ofan extra amount of heme in the plasma resultedin increased concentration of heme-RHBP in thehemolymph (Petretski et al. 1995) and also inthe number of eggs produced (Fig. 10), the needof heme-RHBP for the final composition of ma-ture oocyte becomes evident. Since in the hemo-lymph of the insects, apo-RHBP is alwayspresent and it can be taken up by the oocytes,several possibilities can be considered: (1) apo-RHBP is recycled back to the hemolymph; (2)the apo-RHBP is destroyed inside the oocyte;(3) apo-RHBP is loaded with heme inside theoocyte when heme is available. All these possi-bilities explain why we find only RHBP satu-rated with heme inside the oocyte. In any event,a balanced accumulation of heme-RHBP and Vgseems to be required for egg formation. A pos-sible mechanism to explain these results is thatvesicles containing different proteins are nec-essary for the formation of yolk granules. Onlimiting the vesicles associated with heme-RHBP, the process can not proceed, resultingin a reduction of total number of eggs produced.On the other hand, restoring the levels of heme-RHBP augmented the number of eggs, suggest-ing the need for both proteins for the finalcomposition of oocytes. The mechanism involvedwith this process deserves to be investigated.

ACKNOWLEDGMENTS:

We express our gratitude to José de SouzaLima Junior and José Francisco de Souza Netofor maintaining our colony of Rhodnius prolixus;Lilian S. C. Gomes, Heloisa S. L. Coelho,Rosane O.M.M. Costa, and Sebastião Cruz fortheir excellent technical assistance; Georgia C.Atella, Mario A. Silva-Neto, Gabriela O. P.Silva, and Antonio J. Tempone for helpful sug-gestions; and Katia Calp Gondim for the criti-cal reading of the manuscript. Grants fromConselho Nacional de Desenvolvimento Cientí-fico e Tecnológico (CNPq), Financiadora deEstudos e Projetos (FINEP) and Programa DeApoio ao Desenvolvimento Científico e Tecnoló-gico (PADCT), and Programa de Núcleos deExcelência (PRONEX) supported this work.

142 Machado et al.

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