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Insect Biochemistry and Molecular Biology 31 (2001) 31–40www.elsevier.com/locate/ibmb

Sulfated glycosaminoglycans from ovary ofRhodnius prolixus

Adilson Costa-Filhoa, b, Claudio C. Wernecka, b, Luiz E. Nasciuttic,Hatisaburo Masudab, Georgia C. Atellab, Luiz-Claudio F. Silvaa, b,*

a Laboratório de Tecido Conjuntivo, Hospital Universita´rio Clementino Fraga Filho, Rio de Janeiro, Brazilb Departamento de Bioquı´mica Médica, Instituto de Cieˆncias Biome´dicas, Centro de Cieˆncias da Sau´de, Universidade Federal do Rio de

Janeiro, 21941-590, Caixa Postal 68041, Rio de Janeiro, Brazilc Departamento de Histologia e Embriologia, Instituto de Cieˆncias Biome´dicas, Centro de Cieˆncias da Sau´de, Universidade Federal do Rio de

Janeiro, 21941-590, Caixa Postal 68041, Rio de Janeiro, Brazil

Received 4 January 2000; received in revised form 8 May 2000; accepted 8 May 2000

Abstract

We have characterized sulfated glycosaminoglycans from ovaries of the blood-sucking insectRhodnius prolixus, and determinedparameters of their synthesis and distribution within this organ by biochemical and histochemical procedures. The major sulfatedglycosaminoglycan is heparan sulfate while chondroitin 4–sulfate is a minor component. These glycosaminoglycans are concentratedin the ovarian tissue and are not found inside the oocytes. Besides this, we detected the presence of a sulfated compound dis-tinguished from sulfated glycosaminoglycans and possibly derived from sulfated proteins. Conversely to the compartmental locationof sulfated glycosaminoglycans, the unidentified sulfated compound is located in the ovarian tissue as well as inside the oocytes.Based on these and other findings, the possible roles of ovarian sulfated glycosaminoglycans on the process of oogenesis in theseinsects are discussed. 2001 Elsevier Science Ltd. All rights reserved.

Keywords:Sulfated glycosaminoglycans; Heparan sulfate; Chondroitin sulfate; Sulfated proteins; Oogenesis;Rhodnius prolixus

1. Introduction

Proteoglycans are complex macromolecules that eachcontains a core protein with one or more covalentlybound glycosaminoglycan chains. Glycosaminoglycansare linear polymers of repeating disaccharides that con-tain one hexosamine and either a carboxylated or a sulf-ate ester, or usually both. These molecules can be foundinside cells, on the cell surface and in the extracellularmatrix of a wide variety of vertebrate and invertebratetissues (Cassaro and Dietrich, 1977; Nader et al., 1999).Their strategic location and highly charged nature makethem important biological players in cell–cell and cell–

Abbreviations:α–DGlcUA=α–D4,5–unsaturated glucuronic acid; Gal-NAc=N–acetylated galactosamine; GalNAc(4SO4) andGalNAc(6SO4)=derivatives ofN–acetylated galactosamine bearing asulfate ester at position 4 and at position 6, respectively; HPLC=highpressure liquid chromatography; SAX=strong anion exchange.

* Corresponding author. Address to: Departamento de Bioquı´micaMédica, Instituto de Cieˆncias Biome´dicas. Fax:+55-21-270-8647.

E-mail address:[email protected] (L.-C.F. Silva).

0965-1748/01/$ - see front matter 2001 Elsevier Science Ltd. All rights reserved.PII: S0965-1748 (00)00101-6

matrix interactions that take place during normal andpathological events, related to organogenesis in embry-onic development, cell recognition, adhesion, migration,regulation of growth factor action and lipid metabolism(Gallagher, 1989; Alvarez-Silva et al., 1993; Salmivirtaet al., 1996; Lindahl et al., 1998; Garcia-Abreu et al.,2000).

Insect tissues contain glycosaminoglycans and thepresence of these molecules in various amounts andtypes in different tissues and species has been described(Hoglund, 1976a; Dietrich et al., 1987; Francois, 1989;Cambiazo and Inestrosa, 1990). In particular, Dietrich etal. (1987) have reported that instar nymphs of the hemip-teranRhodnius prolixusactively synthesize sulfated gly-cosaminoglycans. However, qualitative and quantitativebiochemical data on the precise localization and charac-terization of glycosaminoglycans within specific tissuesof these insects are limited.

In order to address this important issue, the presentwork was undertaken to obtain precise information con-cerning the glycosaminoglycans in hemipteran insects.Therefore, we investigated the glycosaminoglycan com-

32 A. Costa-Filho et al. / Insect Biochemistry and Molecular Biology 31 (2001) 31–40

position of isolated ovaries of the blood-sucking hemip-teranRhodnius prolixus. Another aim of this study wasto determine the relative contribution of the ovariantissue and of the oocytes to the synthesis and accumu-lation of the sulfated glycosaminoglycans.

Our results show that ovaries ofR. prolixusproducesulfated compounds corresponding to sulfated glycosam-inoglycans identified as heparan sulfate and chondroitin4–sulfate. In addition, we have demonstrated by bio-chemical and histochemical analysis that these moleculesare concentrated in the ovarian tissue. Besides this, weprovided preliminary evidence to the presence of anotherclass of sulfated compounds distinguished from sulfatedglycosaminoglycans within the ovaries of these insects.To our knowledge, this is the first detailed descriptionof the presence of sulfated compounds, in particular, sul-fated glycosaminoglycans in ovaries of a blood-feed-ing insect.

2. Materials and methods

2.1. Materials

Chondroitin 4–sulfate from whale cartilage, chondro-itin 6–sulfate from shark cartilage, dermatan sulfate frompig skin, and twice-crystallized papain (15 U/mg protein)were purchased from Sigma Chemical Co. (St. Louis,MO, USA). Chondroitin AC lyase (EC 4.2.2.5) fromArthrobacter aurescensand chondroitin ABC lyase (EC4.2.2.4) fromProteus vulgariswere both purchased fromSeikagaku American Inc. (Rockville, MD, USA). Radi-olabeled carrier-free35S–Na2SO4 was obtained fromInstituto de Pesquisas Energe´ticas e Nucleares (Sa˜oPaulo, SP, Brazil). Standard disaccharides for analysisof chondroitin sulfate composition:α–DGlcUA–1→3–GalNAc(4SO4), and α–DGlcUA–1→3–GalNAc(6SO4),were purchased from Seikagaku American Inc.(Rockville, MD).

2.2. Insects

Normal mated females fed with blood at 3-week inter-vals were taken from a colony ofRhodnius prolixusandmaintained at 28°C and 70–80% relative humidity.

2.3. In vivo metabolic labeling of ovarian sulfatedglycosaminoglycans

Two days after a blood meal, insects received aninjection of 20µCi of Na235SO4 at the thorax (Oliveiraet al., 1986), and were kept at 28°C and 70–80% relativehumidity for 24 h. At the end of the labeling period, theinsects were sacrificed and the ovaries were collected,rinsed in PBS and subsequently incubated with 5 vol ofacetone for 24 h at 4°C and dried.

2.4. Isolation of35S–sulfated glycosaminoglycans fromisolated ovaries

Sulfated glycosaminoglycans were isolated from35S–labeled ovaries following the previously describedmethod (Silva et al., 1989). Briefly, dried ovaries weresuspended in sodium acetate buffer, pH 5.5, containing40 mg papain in the presence of 5 mM EDTA and 5mM cysteine at 60°C for 24 h. The suspension was cen-trifuged at 2000g for 10 min at room temperature andthe supernatant, which contained the ovarian glycosami-noglycans, was then applied to a DEAE–cellulose col-umn (3.5×2.5 cm), equilibrated with 0.05 M sodiumacetate (pH 5.0). The column was washed with 100 mlof the same buffer and then eluted step-wise with 25ml of 1.0 M NaCl in the same acetate buffer. The35S–glycosaminoglycans eluted from the column wereexhaustively dialyzed against distilled water, lyophilizedand dissolved in 0.2 ml of distilled water.

2.5. Characterization of the35S–labeledglycosaminoglycans

Radioactive 35S–glycosaminoglycans were charac-terized by anion exchange chromatography on Mono Q–FPLC, agarose gel electrophoresis, digestion with chon-droitin lyases and deaminative cleavage with nitrous acid(Garcia-Abreu et al., 1996; Werneck et al., 1999), asdescribed below.

2.6. Anion-exchange chromatography on mono Q–FPLC

Radiolabeled glycosaminoglycans extracted fromovaries were applied to a Mono Q–FPLC column, equi-librated with 20 mM Tris–HCl (pH 8.0). The columnwas developed by a linear gradient of 0–1.5 M NaCl inthe same buffer. The flow rate of the column was 0.5ml/min, and fractions of 0.5 ml were collected. Theradioactive material was detected by scintillation coun-ting. Two peaks of35S–labeled compounds were elutedfrom the column. The first, Peak F1, eluted with 0.5 MNaCl and the second, Peak F2, eluted with 1.0 M NaCl(see Fig. 1). Fractions corresponding to Peaks F1 andF2 were separately pooled, exhaustively dialyzed againstdistilled water, freeze dried, and stored at220°C.

2.7. Agarose gel electrophoresis

Agarose gel electrophoresis was carried out as pre-viously described (Silva et al., 1992a,b). Approximately10,000 cpm of35S–materials from Peaks F1 and F2 (seeabove), before and after chondroitin lyase digestion ordeaminative cleavage with nitrous acid, as well as a mix-ture of standard chondroitin 4–sulfate, dermatan sulfateand heparan sulfate (10µg of each) were applied to 0.5%

33A. Costa-Filho et al. / Insect Biochemistry and Molecular Biology 31 (2001) 31–40

Fig. 1. Purification of sulfated compounds from isolatedR. prolixusovaries on a Mono Q–FPLC. The DEAE–cellulose–purified sulfatedcompounds were applied to a Mono Q–FPLC and purified as describedunder “Material and Methods”. Fractions were monitored by scintil-lation counting (I). The NaCl concentration in the fractions (- - -) wasdetermined by measuring the conductivity. The fractions correspond-ing to the unidentified sulfated compound (F1) or sulfated GAGs (F2)[cross-hatched peak], as indicated by horizontal bars, were pooled,dialyzed against distilled water and lyophilized. GAGs, glycosaminog-lycans.

agarose gels in 0.05 M 1,3–diaminopropane:acetate (pH9.0). After electrophoresis, glycosaminoglycans werefixed in the gel with 0.1%N–cetyl–N,N,N–trimethylam-monium bromide in water, and stained with 0.1% tol-uidine blue in acetic acid:ethanol:water (0.1:5:5, v/v).The 35S–labeled glycoconjugates were visualized byautoradiography of the stained gels. The radioactivebands having identical electrophoretic migration as stan-dard glycosaminoglycans were carefully scraped into 10ml of 0.5% PPO/toluene solution and counted in a liquidscintillation counter.

2.8. Enzymatic and nitrous acid depolymerization ofthe sulfated compounds

2.8.1. Digestion with chondroitin lyasesDigestions with chondroitin AC or ABC lyases were

carried out according to Saito et al. (1968). Approxi-mately 10,000 cpm of35S–labeled compounds wereincubated with 0.3 units of chondroitin AC lyase orchondroitin ABC lyase for 8 h at 37°C in 100 µl of 50mM Tris–HCl (pH 8.0) containing 5 mM EDTA and 15mM sodium acetate.

2.8.2. Deamination with nitrous acidDeamination by nitrous acid at pH 1.5, was performed

as described by Shively and Conrad (1976). Briefly,approximately 10,000 cpm of35S–compounds wereincubated with 200µl of fresh generated HNO2 at roomtemperature for 10 min. The reaction mixtures were thenneutralized with 1.0 M Na2CO3.

2.9. Analysis of the35S–disaccharides formed byenzymatic depolymerization of ovarian35S–chondroitinsulfate

Purified radiolabeled ovarian glycosaminoglycanchains present in Peak F2 obtained on Mono Q–FPLC(see above) were submitted to exhaustive digestion withchondroitin AC lyase. Disaccharides and chondroitin AClyase-resistant glycosaminoglycans (composed of intactheparan sulfate chains) were recovered by a Superdexpeptide-column (Amersham Pharmacia Biotech) linkedto a HPLC system from Shimadzu (Tokyo, Japan). Thecolumn was eluted with distilled water:acetonitrile:tri-fluoroacetic acid (80:20:0.1, v/v) at a flow rate of 0.5ml/min. Fractions of 0.25 ml were collected, monitoredfor UV absorbance at 232 nm and the radioactivity wascounted in a liquid scintillation counter. Fractions corre-sponding to disaccharides and to the chondroitin AClyase-resistant glycosaminoglycans (eluted at the voidvolume) were pooled, freeze dried, and stored at220°C.

The lyase-derived radiolabeled disaccharides and stan-dard compounds were subjected to a SAX–HPLC ana-lytical column (250×4.6 mm, Sigma–Aldrich), as fol-lows. After equilibration in the mobile phase (distilledwater adjusted to pH 3.5 with HCl) at 0.5 ml/min,samples were injected and disaccharides eluted with alinear gradient of NaCl from 0 to 1.0 M over 45 min inthe same mobile phase. The eluant was collected in 0.5ml fractions and monitored for35S–labeled disaccharidecontent for comparison with lyase derived disaccharidestandards.

2.10. Extraction and analysis of native protein-linked35S–sulfated compounds from ovaries of R. prolixus

As an attempt to isolate native protein-linked35S–sul-fated compounds, fresh collected35S–labeled ovarieswere homogenized in 5 ml of 0.15 M NaCl containing0.05 mg/ml each of soybean trypsin inhibitor, leupeptin,lima bean trypsin inhibitor and antipain, and 1 mMbenzamidine. The homogenate was centrifuged at roomtemperature for 5 min at 10,000g. Pellet(homogenization-resistant material) and supernatant(ovary extract) were separated. The former was incu-bated with 5 vol of acetone for 24 h at 4°C. 35S–sulfatedcompounds were extracted from the dried acetone pow-der by papain digestion as described above.35S–sulfatedcompounds present in the supernatant and thoseextracted from the pellet were analyzed by anion-exchange chromatography on Mono Q–FPLC, followedby agarose gel electrophoresis, as described above.

2.11. Distribution of35S–sulfated compounds betweenovary tissue and oocytes

Fresh-collected35S–labeled ovaries were dissected inorder to separate chorionated oocytes from the ovarian


tissue. Chorionated oocytes were dissected out ofovarian tissues, washed extensively in saline and homo-genized. The remaining tissue was considered ovariantissue, but it also contained non-chorionated oocytes,which were difficult to remove.35S–sulfated compoundswere extracted from the two fractions by papain diges-tion and analyzed by anion exchange chromatographyon Mono Q–FPLC and agarose gel electrophoresis, asdescribed above.

2.12. Histochemical detection of sulfated compoundsin ovaries of R. prolixus

Ovaries and the female reproductive tract from twoanimals were dissected and fixed in 4% paraformal-dehyde in Sorensen phosphate buffer (0.1 M, pH 7.4) at4°C overnight. After fixation and washing, the tissueswere dehydrated in ethanol and embedded in parafin.The sections obtained were stained with the cationic dye1,9–dimethylmethylene blue (Farndale et al., 1986) in0.1 N HCl, containing 0.04 mM glycine and 0.04 NaCl,according to Pava˜o et al. (1994).

3. Results

Sulfated compounds were isolated by papain digestionfrom in vivo metabolically35S–labeled ovaries ofR. pro-lixus. The 35S–sulfated compounds were then analyzedby anion-exchange chromatography and agarose gelelectrophoresis, before and after enzymatic and nitrousacid depolymerization.

3.1. Identification and relative proportions of thevarious sulfated compounds produced by ovaries ofRhodnius prolixus

35S–labeled compounds from ovaries ofR. prolixuswere analyzed by anion-exchange chromatography on aMono Q–FPLC. On elution with a linear gradient ofNaCl, the ovarian compounds showed two35S–labeledcomponents (Fig. 1) designated as F1 and F2, and elutedat 0.5 and 1.0 M NaCl, respectively.

3.2. F2 is a mixture of heparan sulfate andchondroitin 4–sulfate

Further characterization of the35S–glycosaminogly-cans present in F2 by agarose gel electrophoresis (Fig.2B) revealed that the major electrophoretic band had thesame mobility as heparan sulfate standard. It resistedchondroitin AC and ABC lyase digestion, but totally dis-appeared after deaminative cleavage by nitrous acid. Theless intense band had the same mobility as chondroitin4/6 sulfate standard, and totally disappeared from the gelafter digestion with chondroitin AC or ABC lyases (F2

in Fig. 2B). Therefore, the major electrophoretic bandcorresponds to35S–heparan sulfate (75% of totalglycosaminoglycans), while the less intense band ismainly chondroitin sulfate. No dermatan sulfate couldbe detected among glycosaminoglycans isolated fromthe ovary ofR. prolixus.

We further investigated the structure of the ovarianchondroitin sulfate by enzymatic degradation with chon-droitin lyase. The disaccharides formed by exhaustivedigestion of the ovarian35S–labeled glycosaminoglycanswith chondroitin AC lyase were then analyzed on aSAX–HPLC column and the results are shown graphi-cally (Fig. 3). The only product observed wasα–D–GlcUA–GalNAc(4SO4) (Fig. 3B) derived from chondro-itin 4–sulfate (seestandardsin Fig. 3A). Taken together,these results show that the ovary ofR. prolixussynthe-sizes mainly heparan sulfate and small amount of chond-roitin 4–sulfate.

3.3. F1 contains an another class of sulfatedcompound distinguished from sulfatedglycosaminoglycans

The 35S–sulfated compound present in the fraction F1was further analyzed by agarose gel electrophoresis (Fig.2A). Only one band could be observed, with an electro-phoretic mobility between heparan sulfate and dermatansulfate standards. It resisted enzymatic degradation withchondroitin lyases or deaminative cleavage with nitrousacid (F1 in Fig. 2A). These results indicated that F1 doesnot contain sulfated glycosaminoglycans as observed forF2. Possibly this fraction contains a different type of sul-fated compound that may be related to sulfated proteins.

3.4. Isolation of native35S–sulfated compounds fromfresh collected ovaries

In an attempt to obtain native35S–sulfated com-pounds, we homogenized freshly collected and35S–lab-eled ovaries fromR. prolixusin the presence of a cock-tail of protease inhibitors. This procedure solubilizes|50% of the unidentified35S–sulfated compound presentin Peak F1, while the35S–glycosaminoglycans remainentirely in the residue of the homogenization procedure(Fig. 4). The soluble35S–compound and the35S–labeledmolecules extracted from the residue by papain digestionwere analyzed by anion-exchange chromatography onMono Q–FPLC, followed by agarose gel electrophoresis.The native (and soluble) unidentified35S–compoundeluted from the column as a single and sharp peak at aslightly lower concentration of NaCl when comparedwith the unidentified35S–compound obtained by papaindigestion. The soluble35S–compound showed a slightslower mobility on agarose gel when compared with thepapain-extracted molecule (compare Fig. 4C and D).

On the other hand, the results obtained analyzing the


Fig. 2. Autoradiograms of agarose gel electrophoresis of the unidentified sulfated compound (F1) (A) or sulfated GAGs (F2) (B), before (2) andafter enzymatic degradation with chondroitin AC and ABC lyases (+) or deaminative cleavage by nitrous acid (+). The agarose gel electrophoresiswas performed as described under “Material and Methods”. HS=heparan sulfate; DS=dermatan sulfate; CS=chondroitin 4/6 sulfate.

Fig. 3. Anion-exchange HPLC analysis of the disaccharides formedby chondroitin AC lyase digestion of the ovarian radiolabeled GAGs.A mixture of disaccharide standards (A) and the disaccharides formedby exhaustive action of chondroitin AC lyase on the35S–labeledovarian chondroitin sulfate from peak F2 were applied to a 250×4.6mm Spherisorb–SAX column, linked to an HPLC system. The columnwas eluted with a gradient of NaCl as described under “Material andMethods”. The eluant was monitored for UV absorbance at 232 nmand the radioactivity counted in a liquid scintillation counter. The num-bered peaks correspond to the elution positions of known disaccharidestandards as follows: Peak 1,α–DGlcUA–1→3–GlcNAc(6SO4); Peak2 α–DGlcUA–1→3–GalNAc(4SO4).

35S–sulfated materials extracted by papain digestionfrom the residue of the homogenization process showedsimilar chromatographic and electrophoretic patterns asthose obtained when35S–sulfated compounds wereextracted by papain digestion of whole ovaries (compare

Figs. 1, 2, 4B and D, respectively). But, the proportionof the unidentified35S–sulfated compound in the homo-genization-residue has decreased, as expected.

3.5. Distribution of35S–sulfated compounds betweenthe ovarian tissue and the oocytes

35S–sulfated compounds were extracted by papainfrom the ovarian tissue and from the oocyte’s content(see Material and Methods for details on the dissectionprocess). These two materials were analyzed by anion-exchange chromatography on Mono Q–FPLC (Fig. 5).The unidentified sulfated compound was detected inextracts from ovarian tissue and from the oocytes andthe two preparations eluted from Mono Q–FPLC withthe same NaCl concentration. Both preparations exhib-ited the same electrophoretic migration on agarose gelelectrophoresis (Fig. 5C–D). Sulfated glycosaminogly-cans were detected only in the ovarian tissue (cross-hatched peak in Fig. 5A), as confirmed by agarose gelelectrophoresis (F2 in Fig. 5C). No sulfated glycosamin-oglycans were detected in the oocyte’s content fraction(F2 in Fig. 5B and D). These results suggest that theunidentified sulfated compound is found in both theovarian tissue and inside the oocytes. On the contrary,sulfated glycosaminoglycans can be found only in theovarian tissue.

3.6. Metachromatic staining of sulfated materials inovaries of R. prolixus

In order to confirm the biochemical results on the dis-tribution of the sulfated compounds described above, wecarried out the histochemical detection of sulfatedmaterials in whole ovaries by using the cationic dye 1,9–dimethylmethylene blue, which stains (purple color)these compounds (Pava˜o et al., 1994). The metachro-matic staining showed the presence of sulfated com-pounds in the ovarian tissue as well as inside the oocytes(Fig. 6). A strong metachromasia was observed sur-rounding the epithelial surface of the oviduct (Fig. 6C),


Fig. 4. Anion-exchange chromatography of the native unidentifiedsulfated compound extracted by homogenization of whole ovary (A).The 35S–labeled compounds were extracted by homogenization ofwhole ovaries in the presence of protease inhibitors. The extractedmaterial (F1) was analyzed by anion-exchange chromatography on aMono Q–FPLC column. The35S–labeled material that remained in theresidue of the homogenization process (F1 and F2) was extracted bypapain digestion and analyzed in the same column and at the samecondition (B). The ovarian35S–sulfated GAG peak (F2) is cross-hatched. In C and D are shown the autoradiograms of agarose gelelectrophoresis of the native unidentified sulfated compound (C) andof the homogenization-resistant (papain released)35S–sulfated material(D) purified by the anion-exchange chromatography. The agarose gelelectrophoresis was performed as described in Fig. 2.

whereas a less intense one could be seen inside theoocytes in the vitellum (Fig. 6D). More important, a met-achromasia is also observed around the follicle cells(Fig. 6E). Based on the biochemical results showing thatonly the unidentified sulfated compound was present inthe oocyte’s fraction (see Fig. 5B and D) we attributed

Fig. 5. Anion-exchange chromatography of the35S–sulfated com-pounds extracted by papain digestion of the ovarian tissue (A) and ofthe oocyte’s content (B), see “Material and methods” for details onthe dissection process. The extracted materials were analyzed by anion-exchange chromatography on a Mono Q–FPLC column. The ovarian35S–sulfated GAG peak is cross-hatched. In C and D are shown theautoradiograms of agarose gel electrophoresis of the35S–sulfated com-pounds extracted by papain digestion of the ovarian tissue (C) and ofthe oocyte’s content (D) purified by the anion-exchange chromato-graphy. The agarose gel electrophoresis was performed as describedin Fig. 2.

to this compound the metachromasia seen in this com-partment (Fig. 6D). The metachromatic materialobserved around the follicle cells as well as in the ovi-duct (Fig. 6E and C, respectively) may be attributed toboth sulfated glycosaminoglycans and the unidentifiedsulfated compound, since both compounds could bedetected biochemically in the fraction containing


Fig. 6. Light micrographs of theR. prolixusovaries and female reproductive tract (A) stained with the cationic dye 1,9–dimethylmethylene blue.Ovaries and the female reproductive tract from two animals were dissected and fixed in 4% paraformaldehyde in Sorensen phosphate buffer (0.1M, pH 7.4) at 4°C overnight. After fixation and washing, the tissues were dehydrated in ethanol and embedded in parafin. The sections obtainedwere stained with 1,9–dimethylmethylene blue. After staining, a section showing the oviduct (Ov) and a oocyte (O) was examined in an Olympuslight microscope with magnification×100 (B). In (C) and (D) are shown amplification (×200) of the oviduct and of the oocyte, respectively. In (E)is shown, with a great magnification (×1000), a section of the follicle cells (Fc). The surface of the oviduct, the vitellum inside the oocytesand the surface of the follicle cells all display a purple color when stained with 1,9–dimethylmethylene blue (see arrows in panels C, D andE, respectively).

material extracted from the ovarian tissue (see Fig. 5Aand C).

4. Discussion

The process of oogenesis inR. prolixusand in otherinsects is characterized by the rapid accumulation of pro-teins and lipids by the oocytes and a great number ofeggs are produced in a relatively short period of time.This process is very complex and involves several

organs. The eggs ofR. prolixuscontain, in addition tovitellin, which is stored in the form of yolk granules(Oliveira et al., 1986) a variety of other proteins(Oliveira et al., 1995; Silva-Neto et al., 1996) and a largeamount of lipids (Gondim et al., 1989). Thus, the processof egg production requires a continuous supply of pro-teins and lipids. The lipids are transported to the ovaryby the lipoprotein lipophorin, where they are unloaded(Gondim et al., 1989) in association with specific bind-ing sites on the surface of oocytes (Machado et al.,1996). As illustrated above there is a great amount of


information about the presence and role of both proteinand lipids on oogenesis inR. prolixus. In contrast, thesame information regarding sulfated glycoconjugates,specially sulfated glycosaminoglycans, in the ovary ofthese insects and consequently on the process of oogen-esis that takes place in this organ is scarce.

Dietrich et al. (1987) have already reported the pres-ence of sulfated glycosaminoglycans inR. prolixus. Theauthors have characterized, by biochemical methods,sulfated glycosaminoglycans that were extracted by pro-tease digestion from whole instar nymphs ofR. prolixus,and have found the predominant expression of heparansulfate and minor amounts of chondroitin 4–sulfate. Athird sulfated compound was also observed along withthe ovarian sulfated glycosaminoglycans. This materialshowed a slow migration on agarose gel electrophoresisnear to that of the heparan sulfate standard, and resistedenzymatic degradation with both chondroitin lyases andheparan sulfate lyases and it remained unidentified(Dietrich et al., 1987).

We have now focused our studies on glycosaminogly-can composition of a specific organ ofR. prolixus, theovary. Our results show the expression of heparan sulf-ate and chondroitin 4–sulfate in ovaries ofR. prolixus.Heparan sulfate was the major glycosaminoglycan foundin this organ. These compounds could not be extractedby a single homogenization of the whole ovaries. On thecontrary, they remained in the residue of the extractionprocess, suggesting these molecules may have stronginteractions with other components of the microenviron-ment within the ovaries, as already observed for glycosa-minoglycans from mammalian tissues. By using bio-chemical and histochemical methods we were able todetermine the distribution of sulfated glycosaminogly-cans between the ovarian tissue and the oocytes. Ourresults show that sulfated glycosaminoglycans are con-centrated in the ovarian tissue and are absent insidethe oocytes.

Several reports have demonstrated that glycosaminog-lycans are present from the early stage of developmentin insects and that these molecules occur in increasingquantities during the life cycle (Hoglund, 1976b; Fran-cois, 1989; Cambiazo and Inestrosa, 1990). InR. pro-lixus, Dietrich et al. (1987) have suggested that sulfatedglycosaminoglycans may be involved in the process ofmolting. Kelly and Telfer (1979) have demonstrated inHyalophora that the meshwork of spaces that separatethe follicle cells from one another, as well as the per-ioocytic space between the epithelium and the oocyteand between the folds in the oocyte surface, are filledwith a proteoglycan matrix, which was proposed to beable to reversibly hold hemolymph proteins. Huebnerand Anderson (1972) have shown that the follicular epi-thelium on the lateral aspects of the oocyte ofR. prolixusalso undergoes morphological changes eventually cre-ating intercellular spaces. Curiously, they have also dem-

onstrated that sections of vitellogenic follicles presenteda metachromatic material stained by toluidine blue.Therefore, the authors have suggested that endogenouslyproduced proteins may theoretically reach the oocyte ofR. prolixus via the extracellular spaces as they do inHyalophora (Telfer and Anderson, 1968).

4.1. Do the ovarian sulfated glycosaminoglycans playa role on the oogenesis process in R. prolixus?

Our biochemical results showed that sulfated glycosa-minoglycans were present in the ovarian tissue. More-over, by histochemical procedures a metachromaticmaterial could be seen in the surface of the follicle cellsthat seems to be related to these compounds. Therefore,it is tempting to say that our results might be in linewith the mechanism, already suggested by other authors,where exogenous produced proteins may theoreticallyreach the oocyte ofR. prolixus via the extracellularspaces that could be filled with a proteoglycan matrix,which might act as binding agents to hemolymph pro-teins (Huebner and Anderson, 1972). A similar mech-anism was already suggested for Hyalophora (Telfer andAnderson, 1968; Kelly and Telfer, 1979), as describedabove. In addition, it is possible to speculate that theovarian sulfated glycosaminoglycans may help in theprocess of lipid transfer by lipophorin.

In previous works we have reported that in the processof oogenesis inR. prolixus, a major hemolymph lipopro-tein, named lipophorin, mediates the transport of lipidsby taking up phospholipids at the fat body (Atella et al.,1992) and also at the midgut (Atella et al., 1995) anddelivering this to the ovary. In addition, we have demon-strated the presence of specific binding sites for lipopho-rin at the surface of oocytes (Machado et al., 1996). Fur-thermore, the presence of lipoprotein lipases, has alsobeen reported that may be involved on the lipid transportmechanism, within the oocytes of other insect species(Van Antwerpen and Law, 1992). More important,Schulz et al. (1991) have demonstrated that the in vitrobinding of locust high-density lipophorin to fat body pro-teins can be inhibited by heparin, a highly sulfated gly-cosaminoglycan. Since interactions among different pro-teins and sulfated glycosaminoglycans, specially heparansulfate, have been reported to depend on their fine struc-ture, additional experiments are necessary to completelycharacterize the structure ofR. prolixus ovarianheparan sulfate.

The presence of metachromatic material, possibly sul-fated glycosaminoglycans, in the surface of the oviductsuggests that these compounds may be an importantstructural component of the ovarian microenvironment.Several experiments are yet necessary to establish theabove mentioned possible roles forR. prolixusovariansulfated glycosaminoglycans. These studies are under


consideration by our group and consist of an importantperspective opened by the present work.

Another important aspect of our present work con-cerns the presence of the unidentified sulfated compoundisolated from the ovaries ofR. prolixusalong with thesulfated glycosaminoglycans. Dietrich et al. (1987) havealso isolated from whole nymphs ofR. prolixusa sul-fated compound unrelated to sulfated glycosaminogly-cans, but they did not make any attempt to fully identifythis compound. In the present work, our results suggestthat the ovarian sulfated compound may possibly bederived from ovarian sulfated proteins. This compoundcould be partially extracted from whole ovaries byhomogenization, as a native protein-linked sulfated com-pound. More important, it could be detected biochemi-cally and histochemically in the ovarian tissue as wellas inside the oocytes. Fausto et al. (1998) have reportedthe presence of sulfated proteins in the ovaries of thestick insectCarausius morosus. Therefore, the completecharacterization of the unidentified sulfated compoundpresent in F1 is another important aspect that deservesfuture investigation.

In conclusion, we believe that the present workdescribing the presence of these sulfated compounds inR. prolixusovaries is timely and worthwhile, by actingas a valuable starting point for further investigation ofpotential roles of sulfated glycosaminoglycans ininsect oogenesis.

Acknowledgements

This work was supported by Conselho Nacional deDesenvolvimento Cientı´fico e Tecnolo´gico (CNPq:PADCT and PRONEX), Fundação de Amparo a` Pes-quisa do Estado do Rio de Janeiro (FAPERJ) and Finan-ciadora de Estudos e Projetos (FINEP). We are gratefulto Adriana A. Piquet for technical assistance.

References

Alvarez-Silva, M., Silva, L.C.F., Borojevic, R., 1993. Cell membrane-associated proteoglycans mediate extramedullar myeloid prolifer-ation in granulomatous inflammatory reactions to schistosome eggs.Journal of Cell Science 104, 477–484.

Atella, G.C., Gondim, K.C., Masuda, H., 1992. Transfer of phospholip-ids from fat body to lipophorin inRhodnius prolixus. Archives ofInsect Biochemistry and Physiology 19, 133–144.

Atella, G.C., Gondim, K.C., Masuda, H., 1995. Loading of lipophorinparticles with phospholipids at the midgut ofRhodnius prolixus.Archives of Insect Biochemistry and Physiology 30, 337–350.

Cambiazo, V., Inestrosa, N.C., 1990. Proteoglycan production inDro-sophilaegg development: effect ofβ–D–xyloside on proteoglycansynthesis and larvae motility. Comparative Biochemistry andPhysiology B 97, 307–314.

Cassaro, C.M.F., Dietrich, C.P., 1977. The distribution of sulfatedmucopolysaccharides in invertebrates. The Journal BiologicalChemistry 252, 2254–2261.

Dietrich, C.P., Nader, H.B., Toma, L., Azambuja, P., Garcia, E.S.,1987. A relationship between the inhibition of heparan sulfate andchondroitin sulfate synthesis and the inhibition of molting by selen-ate in the hemipteranRhodnius prolixus. Biochemical and Biophys-ical Research Communications 146, 652–658.

Farndale, R.W., Buttle, D.J., Barret, A.J., 1986. Improved quantitationand discrimination of sulfated glycosaminoglycans by use of dime-thylmethylene blue. Biochimica et Biophysica Acta 883, 173–177.

Fausto, A.M., Fava, E., Mazzini, M., Cecchettini, A., Giorgi, F., 1998.Confocal scanning laser microscopy of the follicular epithelium inovarioles of the stick insectCarausius morosus. Cell and TissueResearch 293, 551–561.

Francois, J., 1989. The glycosaminoglycans of midgut connectivesheath during development of the mealwormTenebrio molitorL.Comparative Biochemistry and Physiology B 93, 93–98.

Gallagher, J.T., 1989. The extended family of proteoglycans: socialresidents of the pericellular zone. Current Opinion on Cell Biology1, 1201–1218.

Garcia-Abreu, J., Silva, L.C.F., Tovar, F.F., Onofre, G.R., Cavalcante,L.A., Moura Neto, V., 1996. Compartmental distribution of Sul-fated glycosaminoglycans in lateral and medial midbrain astrogliacultures. Glia 17, 339–344.

Garcia-Abreu, J., Mendes, F.A., Onofre, G.R., Freitas, M.S., Silva,L.C.F., Moura Neto, V., Cavalcante, L.A., 2000. Contribution ofheparan sulfate to the non-permissive role of the midline glia tothe growth of midbrain neurites. Glia 29, 260–272.

Gondim, K.C., Oliveira, P.L., Masuda, H., 1989. Lipophorin and oog-enesis inRhodnius prolixus: transfer of phospholipids. Journal ofInsect Physiology 35, 19–27.

Hoglund, L., 1976a. The comparative biochemistry of invertebratemucopolysaccharides. V: Insecta (Calliphora erythrocephala).Comparative Biochemistry and Physiology B 53, 9–14.

Hoglund, L., 1976b. Changes in acid mucopolysaccharides during thedevelopment of the blowflyCalliphora erythrocephala. Journal ofInsect Physiology 22, 917–924.

Huebner, E., Anderson, E., 1972. A cytological study of the ovary ofRhodnius prolixus. I. The ontogeny of the follicular epithelium.Journal of Morphology 136, 459–494.

Kelly, T.J., Telfer, W.H., 1979. The function of the follicular epi-thelium in vitellogenic oncopeltus follicules. Tissue and Cell 11,663–672.

Lindahl, U., Gullberg-Kusche, M., Kjeleń, L., 1998. Regulated diver-sity of heparan sulfate. The Journal Biological Chemistry 273,24979–24982.

Machado, E.A., Atella, G.C., Gondim, K.C., De-Souza, W., Masuda,H., 1996. Characterization and immunocytochemical localizationof lipophorin binding sites in the oocytes ofRhodnius prolixus.Archives of Insect Biochemistry and Physiology 31, 185–196.

Nader, H.B., Chavante, S.F., Dos-Santos, E.A., Oliveira, F.W., De-Paiva, J.F., Jeroˆnimo, S.M.B., Medeiros, G.F., De-Abreu, L.R.D.,Leite, E.L., De-Souza-Filho, J.F., Castro, R.A.B., Toma, L., Tersa-riol, I.L.S., Porcionatto, M.A., Dietrich, C.P., 1999. Heparan sul-fates and heparins: similar compounds performing the same func-tions in vertebrates and invertebrates? Brazilian Journal of Medicaland Biological Research 32, 529–538.

Oliveira, P.L., Gondim, K.C., Guedes, D.M., Masuda, H., 1986.Uptake of yolk proteins inRhodnius prolixus. Journal of InsectPhysiology 32, 859–866.

Oliveira, P.L., Kawooya, J.K., Ribeiro, J.M.C., Meyer, T., Poorman,R., Alves, E.W., Walker, F.A., Machado, E.A., Nussenzveig, R.H.,Padovan, G.J., Masuda, H., 1995. A heme-binding protein fromhemolymph and oocytes of the blood-sucking insectRhodnius pro-lixus. The Journal of Biological Chemistry 270, 10897–10901.

Pavão, M.S.G., Lambert, C.C., Lambert, G., Mouraõ, P.A.S., 1994.Comparison between the sulfated polysaccharides from larval andadult ascidians. Journal Experimental Zoology 269, 89–94.

Saito, N., Yamagata, T., Suzuki, S., 1968. Enzymatic methods for the


determination of small quantities of isomeric chondroitin sulfates.The Journal of Biological Chemistry 243, 1536–1544.

Salmivirta, M., Lidholt, K., Lindahl, U., 1996. Heparan sulfate: a pieceof information. FASEB Journal 10, 1270–1279.

Schulz, T.K., Van der Horst, D.J., Beenakkers, A.M., 1991. Bindingof locust high-density lipophorin to fat body proteins monitored byan enzyme-linked immunosorbant assay. Biological and ChemistryHoppe-Seyler 372, 5–12.

Shively, J.E., Conrad, H.E., 1976. Formation of anhydrosugars in thechemical depolymerization of heparin. Biochemistry 15, 3932–3942.

Silva, L.C.F., Borojevic, R., Mouraõ, P.A.S., 1992a. Membrane-asso-ciated and secreted proteoglycans from a continuous cell linederived from fibrotic schistosomal granulomas. Biochimica etBiophysica Acta 1138, 133–142.

Silva, L.C.F., Borojevic, R., Mouraõ, P.A.S., 1992b. Proteoglycanssynthesized by the hepatic granulomas isolated from schistosome-infected mice and by the granuloma derived connective tissue celllines. Biochimica et Biophysica Acta 1139, 96–104.

Silva, L.C.F., Mouraõ, P.A.S., Borojevic, R., 1989. Patterns of sulfatedglycosaminoglycan synthesis and accumulation in hepatic granu-lomas induced by schistosomal infection. Experimental and Mol-ecular Pathology 50, 411–420.

Silva-Neto, M.A.C., Atella, G.C., Fialho, E., Paes, M.C., Zingali, R.B.,Petretski, J.H., Alves, E.W., Masuda, H., 1996. Isolation of a cal-cium-binding phosphoprotein from the oocytes and hemolymph ofthe blood-sucking insectRhodnius prolixus. The Journal BiologicalChemistry 271, 30227–30232.

Telfer, W.H., Anderson, L.M., 1968. Functional transformationsaccompanying the initiation of a terminal growth phase in the mothoocyte. Developmental Biology 5, 512–535.

Van Antwerpen, R., Law, J.H., 1992. Lipophorin lipase from the yolkof Manduca sextaeggs: identification and partial characterization.Archives of Insect Biochemistry and Physiology 20, 1–12.

Werneck, C.C., Oliveira-Dos-Santos, A.J., Silva, L.C.F., Villa-Verde,D.M.S., Savino, W., Mouraõ, P.A.S., 1999. Thymic epithelial cellssynthesized a heparan sulfate with highly sulfated region. Journalof Cellular Physiology 178, 51–62.

sulfated glycosaminoglycans from ovary of rhodnius prolixusdirectory.umm.ac.id/data...

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