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Sulfated glycosaminoglycans from ovary of Rhodnius prolixus
Adilson Costa-Filho
a, b, Claudio C. Werneck
a, b, Luiz E. Nasciutti
c,
Hatisaburo Masuda
b, Georgia C. Atella
b, Luiz-Claudio F. Silva
a, b,*aLaborato´rio de Tecido Conjuntivo, Hospital Universita´rio Clementino Fraga Filho, Rio de Janeiro, Brazil
bDepartamento de Bioquı´mica Me´dica, 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
cDepartamento 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 insect Rhodnius prolixus, and determined parameters of their synthesis and distribution within this organ by biochemical and histochemical procedures. The major sulfated glycosaminoglycan is heparan sulfate while chondroitin 4–sulfate is a minor component. These glycosaminoglycans are concentrated in 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 location of 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 these insects 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 each contains a core protein with one or more covalently bound glycosaminoglycan chains. Glycosaminoglycans are 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 found inside cells, on the cell surface and in the extracellular matrix of a wide variety of vertebrate and invertebrate tissues (Cassaro and Dietrich, 1977; Nader et al., 1999). Their strategic location and highly charged nature make them important biological players in cell–cell and cell–
Abbreviations:α–DGlcUA=α–D4,5–unsaturated glucuronic acid;
Gal-NAc=N–acetylated galactosamine; GalNAc(4SO4) and
GalNAc(6SO4)=derivatives of N–acetylated galactosamine bearing a
sulfate ester at position 4 and at position 6, respectively; HPLC=high pressure liquid chromatography; SAX=strong anion exchange.
* Corresponding author. Address to: Departamento de Bioquı´mica Me´dica, Instituto de Cieˆncias Biome´dicas. Fax:+55-21-270-8647.
E-mail address: [email protected] (L.-C.F. Silva).
0965-1748/01/$ - see front matter2001 Elsevier Science Ltd. All rights reserved. PII: S 0 9 6 5 - 1 7 4 8 ( 0 0 ) 0 0 1 0 1 - 6
matrix interactions that take place during normal and pathological 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; Salmivirta et al., 1996; Lindahl et al., 1998; Garcia-Abreu et al., 2000).
Insect tissues contain glycosaminoglycans and the presence of these molecules in various amounts and types in different tissues and species has been described (Hoglund, 1976a; Dietrich et al., 1987; Francois, 1989; Cambiazo and Inestrosa, 1990). In particular, Dietrich et al. (1987) have reported that instar nymphs of the hemip-teran Rhodnius prolixus actively synthesize sulfated gly-cosaminoglycans. However, qualitative and quantitative biochemical data on the precise localization and charac-terization of glycosaminoglycans within specific tissues of these insects are limited.
com-position of isolated ovaries of the blood-sucking hemip-teran Rhodnius prolixus. Another aim of this study was to determine the relative contribution of the ovarian tissue and of the oocytes to the synthesis and accumu-lation of the sulfated glycosaminoglycans.
Our results show that ovaries of R. prolixus produce sulfated compounds corresponding to sulfated glycosam-inoglycans identified as heparan sulfate and chondroitin 4–sulfate. In addition, we have demonstrated by bio-chemical and histobio-chemical analysis that these molecules are concentrated in the ovarian tissue. Besides this, we provided preliminary evidence to the presence of another class of sulfated compounds distinguished from sulfated glycosaminoglycans within the ovaries of these insects. To our knowledge, this is the first detailed description of 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 from pig 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) from Arthrobacter aurescens and chondroitin ABC lyase (EC 4.2.2.4) from Proteus vulgaris were both purchased from Seikagaku American Inc. (Rockville, MD, USA). Radi-olabeled carrier-free 35S–Na
2SO4 was obtained from
Instituto de Pesquisas Energe´ticas e Nucleares (Sa˜o Paulo, SP, Brazil). Standard disaccharides for analysis of 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 of Rhodnius prolixus and maintained at 28°C and 70–80% relative humidity.
2.3. In vivo metabolic labeling of ovarian sulfated glycosaminoglycans
Two days after a blood meal, insects received an injection of 20 µCi of Na235SO4 at the thorax (Oliveira
et al., 1986), and were kept at 28°C and 70–80% relative humidity for 24 h. At the end of the labeling period, the insects were sacrificed and the ovaries were collected, rinsed in PBS and subsequently incubated with 5 vol of acetone for 24 h at 4°C and dried.
2.4. Isolation of 35S–sulfated glycosaminoglycans from isolated ovaries
Sulfated glycosaminoglycans were isolated from35S–
labeled ovaries following the previously described method (Silva et al., 1989). Briefly, dried ovaries were suspended in sodium acetate buffer, pH 5.5, containing 40 mg papain in the presence of 5 mM EDTA and 5 mM cysteine at 60°C for 24 h. The suspension was cen-trifuged at 2000g for 10 min at room temperature and the 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 sodium acetate (pH 5.0). The column was washed with 100 ml of the same buffer and then eluted step-wise with 25 ml of 1.0 M NaCl in the same acetate buffer. The 35S–
glycosaminoglycans eluted from the column were exhaustively dialyzed against distilled water, lyophilized and dissolved in 0.2 ml of distilled water.
2.5. Characterization of the 35S–labeled glycosaminoglycans
Radioactive 35
S–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), as described below.
2.6. Anion-exchange chromatography on mono Q– FPLC
Radiolabeled glycosaminoglycans extracted from ovaries were applied to a Mono Q–FPLC column, equi-librated with 20 mM Tris–HCl (pH 8.0). The column was developed by a linear gradient of 0–1.5 M NaCl in the same buffer. The flow rate of the column was 0.5 ml/min, and fractions of 0.5 ml were collected. The radioactive material was detected by scintillation coun-ting. Two peaks of 35
S–labeled compounds were eluted from the column. The first, Peak F1, eluted with 0.5 M NaCl and the second, Peak F2, eluted with 1.0 M NaCl (see Fig. 1). Fractions corresponding to Peaks F1 and F2 were separately pooled, exhaustively dialyzed against distilled water, freeze dried, and stored at 220°C.
2.7. Agarose gel electrophoresis
Agarose gel electrophoresis was carried out as pre-viously described (Silva et al., 1992a,b). Approximately 10,000 cpm of35S–materials from Peaks F1 and F2 (see
Fig. 1. Purification of sulfated compounds from isolated R. prolixus ovaries on a Mono Q–FPLC. The DEAE–cellulose–purified sulfated compounds were applied to a Mono Q–FPLC and purified as described under “Material and Methods”. Fractions were monitored by scintil-lation counting (I). The NaCl concentration in the fractions (- - -) was determined 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 (pH 9.0). After electrophoresis, glycosaminoglycans were fixed 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 by
autoradiography of the stained gels. The radioactive bands having identical electrophoretic migration as stan-dard glycosaminoglycans were carefully scraped into 10 ml of 0.5% PPO/toluene solution and counted in a liquid scintillation counter.
2.8. Enzymatic and nitrous acid depolymerization of the sulfated compounds
2.8.1. Digestion with chondroitin lyases
Digestions with chondroitin AC or ABC lyases were carried out according to Saito et al. (1968). Approxi-mately 10,000 cpm of 35S–labeled compounds were
incubated with 0.3 units of chondroitin AC lyase or chondroitin ABC lyase for 8 h at 37°C in 100µl of 50 mM Tris–HCl (pH 8.0) containing 5 mM EDTA and 15 mM sodium acetate.
2.8.2. Deamination with nitrous acid
Deamination by nitrous acid at pH 1.5, was performed as described by Shively and Conrad (1976). Briefly, approximately 10,000 cpm of 35S–compounds were
incubated with 200µl of fresh generated HNO2at room
temperature for 10 min. The reaction mixtures were then neutralized with 1.0 M Na2CO3.
2.9. Analysis of the 35S–disaccharides formed by enzymatic depolymerization of ovarian 35S–chondroitin sulfate
Purified radiolabeled ovarian glycosaminoglycan chains present in Peak F2 obtained on Mono Q–FPLC (see above) were submitted to exhaustive digestion with chondroitin AC lyase. Disaccharides and chondroitin AC lyase-resistant glycosaminoglycans (composed of intact heparan sulfate chains) were recovered by a Superdex peptide-column (Amersham Pharmacia Biotech) linked to a HPLC system from Shimadzu (Tokyo, Japan). The column was eluted with distilled water:acetonitrile:tri-fluoroacetic acid (80:20:0.1, v/v) at a flow rate of 0.5 ml/min. Fractions of 0.25 ml were collected, monitored for UV absorbance at 232 nm and the radioactivity was counted in a liquid scintillation counter. Fractions corre-sponding to disaccharides and to the chondroitin AC lyase-resistant glycosaminoglycans (eluted at the void volume) 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 (distilled water adjusted to pH 3.5 with HCl) at 0.5 ml/min, samples were injected and disaccharides eluted with a linear gradient of NaCl from 0 to 1.0 M over 45 min in the same mobile phase. The eluant was collected in 0.5 ml fractions and monitored for35S–labeled disaccharide
content for comparison with lyase derived disaccharide standards.
2.10. Extraction and analysis of native protein-linked 35S–sulfated compounds from ovaries of R. prolixus
As an attempt to isolate native protein-linked35
S–sul-fated compounds, fresh collected 35S–labeled ovaries
were homogenized in 5 ml of 0.15 M NaCl containing 0.05 mg/ml each of soybean trypsin inhibitor, leupeptin, lima bean trypsin inhibitor and antipain, and 1 mM benzamidine. The homogenate was centrifuged at room temperature 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.35
S–sulfated compounds were extracted from the dried acetone pow-der by papain digestion as described above.35S–sulfated
compounds present in the supernatant and those extracted from the pellet were analyzed by anion-exchange chromatography on Mono Q–FPLC, followed by agarose gel electrophoresis, as described above.
2.11. Distribution of 35S–sulfated compounds between ovary tissue and oocytes
Fresh-collected35
tissue. Chorionated oocytes were dissected out of ovarian tissues, washed extensively in saline and homo-genized. The remaining tissue was considered ovarian tissue, but it also contained non-chorionated oocytes, which were difficult to remove.35S–sulfated compounds
were extracted from the two fractions by papain diges-tion and analyzed by anion exchange chromatography on Mono Q–FPLC and agarose gel electrophoresis, as described above.
2.12. Histochemical detection of sulfated compounds in ovaries of R. prolixus
Ovaries and the female reproductive tract from two animals were dissected and fixed in 4% paraformal-dehyde in Sorensen phosphate buffer (0.1 M, pH 7.4) at 4°C overnight. After fixation and washing, the tissues were dehydrated in ethanol and embedded in parafin. The sections obtained were stained with the cationic dye 1,9–dimethylmethylene blue (Farndale et al., 1986) in 0.1 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 digestion from in vivo metabolically35S–labeled ovaries of R.
pro-lixus. The 35S–sulfated compounds were then analyzed
by anion-exchange chromatography and agarose gel electrophoresis, before and after enzymatic and nitrous acid depolymerization.
3.1. Identification and relative proportions of the various sulfated compounds produced by ovaries of Rhodnius prolixus
35S–labeled compounds from ovaries of R. prolixus
were analyzed by anion-exchange chromatography on a Mono Q–FPLC. On elution with a linear gradient of NaCl, the ovarian compounds showed two 35
S–labeled components (Fig. 1) designated as F1 and F2, and eluted at 0.5 and 1.0 M NaCl, respectively.
3.2. F2 is a mixture of heparan sulfate and chondroitin 4–sulfate
Further characterization of the 35
S–glycosaminogly-cans present in F2 by agarose gel electrophoresis (Fig. 2B) revealed that the major electrophoretic band had the same mobility as heparan sulfate standard. It resisted chondroitin AC and ABC lyase digestion, but totally dis-appeared after deaminative cleavage by nitrous acid. The less intense band had the same mobility as chondroitin 4/6 sulfate standard, and totally disappeared from the gel after digestion with chondroitin AC or ABC lyases (F2
in Fig. 2B). Therefore, the major electrophoretic band corresponds to 35S–heparan sulfate (75% of total
glycosaminoglycans), while the less intense band is mainly chondroitin sulfate. No dermatan sulfate could be detected among glycosaminoglycans isolated from the ovary of R. prolixus.
We further investigated the structure of the ovarian chondroitin sulfate by enzymatic degradation with chon-droitin lyase. The disaccharides formed by exhaustive digestion of the ovarian35
S–labeled glycosaminoglycans with chondroitin AC lyase were then analyzed on a SAX–HPLC column and the results are shown graphi-cally (Fig. 3). The only product observed was α–D– GlcUA–GalNAc(4SO4) (Fig. 3B) derived from
chondro-itin 4–sulfate (see standards in Fig. 3A). Taken together, these results show that the ovary of R. prolixus synthe-sizes mainly heparan sulfate and small amount of chond-roitin 4–sulfate.
3.3. F1 contains an another class of sulfated compound distinguished from sulfated glycosaminoglycans
The35
S–sulfated compound present in the fraction F1 was further analyzed by agarose gel electrophoresis (Fig. 2A). Only one band could be observed, with an electro-phoretic mobility between heparan sulfate and dermatan sulfate standards. It resisted enzymatic degradation with chondroitin lyases or deaminative cleavage with nitrous acid (F1 in Fig. 2A). These results indicated that F1 does not contain sulfated glycosaminoglycans as observed for F2. Possibly this fraction contains a different type of sul-fated compound that may be related to sulsul-fated proteins.
3.4. Isolation of native 35S–sulfated compounds from fresh collected ovaries
In an attempt to obtain native 35S–sulfated
com-pounds, we homogenized freshly collected and 35
S–lab-eled ovaries from R. prolixus in the presence of a cock-tail of protease inhibitors. This procedure solubilizes
|50% of the unidentified35S–sulfated compound present
in Peak F1, while the 35S–glycosaminoglycans remain
entirely in the residue of the homogenization procedure (Fig. 4). The soluble35S–compound and the35S–labeled
molecules extracted from the residue by papain digestion were analyzed by anion-exchange chromatography on Mono Q–FPLC, followed by agarose gel electrophoresis. The native (and soluble) unidentified 35S–compound
eluted from the column as a single and sharp peak at a slightly lower concentration of NaCl when compared with the unidentified 35S–compound obtained by papain
digestion. The soluble 35S–compound showed a slight
slower mobility on agarose gel when compared with the papain-extracted molecule (compare Fig. 4C and D).
Fig. 2. Autoradiograms of agarose gel electrophoresis of the unidentified sulfated compound (F1) (A) or sulfated GAGs (F2) (B), before (2) and after enzymatic degradation with chondroitin AC and ABC lyases (+) or deaminative cleavage by nitrous acid (+). The agarose gel electrophoresis was 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 formed by chondroitin AC lyase digestion of the ovarian radiolabeled GAGs. A mixture of disaccharide standards (A) and the disaccharides formed by exhaustive action of chondroitin AC lyase on the 35S–labeled
ovarian chondroitin sulfate from peak F2 were applied to a 250×4.6 mm Spherisorb–SAX column, linked to an HPLC system. The column was eluted with a gradient of NaCl as described under “Material and Methods”. The eluant was monitored for UV absorbance at 232 nm and the radioactivity counted in a liquid scintillation counter. The num-bered peaks correspond to the elution positions of known disaccharide standards as follows: Peak 1,α–DGlcUA–1→3–GlcNAc(6SO4); Peak
2α–DGlcUA–1→3–GalNAc(4SO4).
35S–sulfated materials extracted by papain digestion
from the residue of the homogenization process showed similar chromatographic and electrophoretic patterns as those obtained when 35
S–sulfated compounds were extracted by papain digestion of whole ovaries (compare
Figs. 1, 2, 4B and D, respectively). But, the proportion of the unidentified 35S–sulfated compound in the
homo-genization-residue has decreased, as expected.
3.5. Distribution of 35S–sulfated compounds between the ovarian tissue and the oocytes
35S–sulfated compounds were extracted by papain
from the ovarian tissue and from the oocyte’s content (see Material and Methods for details on the dissection process). These two materials were analyzed by anion-exchange chromatography on Mono Q–FPLC (Fig. 5). The unidentified sulfated compound was detected in extracts from ovarian tissue and from the oocytes and the two preparations eluted from Mono Q–FPLC with the same NaCl concentration. Both preparations exhib-ited the same electrophoretic migration on agarose gel electrophoresis (Fig. 5C–D). Sulfated glycosaminogly-cans were detected only in the ovarian tissue (cross-hatched peak in Fig. 5A), as confirmed by agarose gel electrophoresis (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 the unidentified sulfated compound is found in both the ovarian tissue and inside the oocytes. On the contrary, sulfated glycosaminoglycans can be found only in the ovarian tissue.
3.6. Metachromatic staining of sulfated materials in ovaries of R. prolixus
Fig. 4. Anion-exchange chromatography of the native unidentified sulfated compound extracted by homogenization of whole ovary (A). The 35S–labeled compounds were extracted by homogenization of
whole ovaries in the presence of protease inhibitors. The extracted material (F1) was analyzed by anion-exchange chromatography on a Mono Q–FPLC column. The35S–labeled material that remained in the
residue of the homogenization process (F1 and F2) was extracted by papain digestion and analyzed in the same column and at the same condition (B). The ovarian 35S–sulfated GAG peak (F2) is
cross-hatched. In C and D are shown the autoradiograms of agarose gel electrophoresis of the native unidentified sulfated compound (C) and of the homogenization-resistant (papain released)35S–sulfated material
(D) purified by the anion-exchange chromatography. The agarose gel electrophoresis was performed as described in Fig. 2.
whereas a less intense one could be seen inside the oocytes 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 that only the unidentified sulfated compound was present in the oocyte’s fraction (see Fig. 5B and D) we attributed
Fig. 5. Anion-exchange chromatography of the 35S–sulfated
com-pounds extracted by papain digestion of the ovarian tissue (A) and of the oocyte’s content (B), see “Material and methods” for details on the dissection process. The extracted materials were analyzed by anion-exchange chromatography on a Mono Q–FPLC column. The ovarian
35S–sulfated GAG peak is cross-hatched. In C and D are shown the
autoradiograms of agarose gel electrophoresis of the35S–sulfated
com-pounds extracted by papain digestion of the ovarian tissue (C) and of the oocyte’s content (D) purified by the anion-exchange chromato-graphy. The agarose gel electrophoresis was performed as described in Fig. 2.
Fig. 6. Light micrographs of the R. prolixus ovaries 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 (0.1 M, pH 7.4) at 4°C overnight. After fixation and washing, the tissues were dehydrated in ethanol and embedded in parafin. The sections obtained were stained with 1,9–dimethylmethylene blue. After staining, a section showing the oviduct (Ov) and a oocyte (O) was examined in an Olympus light 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 oocytes and the surface of the follicle cells all display a purple color when stained with 1,9–dimethylmethylene blue (see arrows in panels C, D and E, respectively).
material extracted from the ovarian tissue (see Fig. 5A and C).
4. Discussion
The process of oogenesis in R. prolixus and in other insects is characterized by the rapid accumulation of pro-teins and lipids by the oocytes and a great number of eggs are produced in a relatively short period of time. This process is very complex and involves several
information about the presence and role of both protein and lipids on oogenesis in R. prolixus. In contrast, the same information regarding sulfated glycoconjugates, specially sulfated glycosaminoglycans, in the ovary of these 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 in R. prolixus. The authors have characterized, by biochemical methods, sulfated glycosaminoglycans that were extracted by pro-tease digestion from whole instar nymphs of R. prolixus, and have found the predominant expression of heparan sulfate and minor amounts of chondroitin 4–sulfate. A third sulfated compound was also observed along with the ovarian sulfated glycosaminoglycans. This material showed a slow migration on agarose gel electrophoresis near to that of the heparan sulfate standard, and resisted enzymatic degradation with both chondroitin lyases and heparan sulfate lyases and it remained unidentified (Dietrich et al., 1987).
We have now focused our studies on glycosaminogly-can composition of a specific organ of R. prolixus, the ovary. Our results show the expression of heparan sulf-ate and chondroitin 4–sulfsulf-ate in ovaries of R. prolixus. Heparan sulfate was the major glycosaminoglycan found in this organ. These compounds could not be extracted by a single homogenization of the whole ovaries. On the contrary, they remained in the residue of the extraction process, suggesting these molecules may have strong interactions with other components of the microenviron-ment within the ovaries, as already observed for glycosa-minoglycans from mammalian tissues. By using bio-chemical and histobio-chemical methods we were able to determine the distribution of sulfated glycosaminogly-cans between the ovarian tissue and the oocytes. Our results show that sulfated glycosaminoglycans are con-centrated in the ovarian tissue and are absent inside the oocytes.
Several reports have demonstrated that glycosaminog-lycans are present from the early stage of development in insects and that these molecules occur in increasing quantities during the life cycle (Hoglund, 1976b; Fran-cois, 1989; Cambiazo and Inestrosa, 1990). In R. pro-lixus, Dietrich et al. (1987) have suggested that sulfated glycosaminoglycans may be involved in the process of molting. Kelly and Telfer (1979) have demonstrated in Hyalophora that the meshwork of spaces that separate the follicle cells from one another, as well as the per-ioocytic space between the epithelium and the oocyte and between the folds in the oocyte surface, are filled with a proteoglycan matrix, which was proposed to be able to reversibly hold hemolymph proteins. Huebner and Anderson (1972) have shown that the follicular epi-thelium on the lateral aspects of the oocyte of R. prolixus also undergoes morphological changes eventually cre-ating intercellular spaces. Curiously, they have also
dem-onstrated that sections of vitellogenic follicles presented a metachromatic material stained by toluidine blue. Therefore, the authors have suggested that endogenously produced proteins may theoretically reach the oocyte of R. prolixus via the extracellular spaces as they do in Hyalophora (Telfer and Anderson, 1968).
4.1. Do the ovarian sulfated glycosaminoglycans play a 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 metachromatic material could be seen in the surface of the follicle cells that seems to be related to these compounds. Therefore, it is tempting to say that our results might be in line with the mechanism, already suggested by other authors, where exogenous produced proteins may theoretically reach the oocyte of R. prolixus via the extracellular spaces 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 and Anderson, 1968; Kelly and Telfer, 1979), as described above. In addition, it is possible to speculate that the ovarian sulfated glycosaminoglycans may help in the process of lipid transfer by lipophorin.
In previous works we have reported that in the process of oogenesis in R. prolixus, a major hemolymph lipopro-tein, named lipophorin, mediates the transport of lipids by taking up phospholipids at the fat body (Atella et al., 1992) and also at the midgut (Atella et al., 1995) and delivering 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 also been reported that may be involved on the lipid transport mechanism, within the oocytes of other insect species (Van Antwerpen and Law, 1992). More important, Schulz et al. (1991) have demonstrated that the in vitro binding 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 heparan sulfate, have been reported to depend on their fine struc-ture, additional experiments are necessary to completely characterize the structure of R. prolixus ovarian heparan sulfate.
consideration by our group and consist of an important perspective opened by the present work.
Another important aspect of our present work con-cerns the presence of the unidentified sulfated compound isolated from the ovaries of R. prolixus along with the sulfated glycosaminoglycans. Dietrich et al. (1987) have also isolated from whole nymphs of R. prolixus a sul-fated compound unrelated to sulsul-fated glycosaminogly-cans, but they did not make any attempt to fully identify this compound. In the present work, our results suggest that the ovarian sulfated compound may possibly be derived from ovarian sulfated proteins. This compound could be partially extracted from whole ovaries by homogenization, as a native protein-linked sulfated com-pound. More important, it could be detected biochemi-cally and histochemibiochemi-cally in the ovarian tissue as well as inside the oocytes. Fausto et al. (1998) have reported the presence of sulfated proteins in the ovaries of the stick insect Carausius morosus. Therefore, the complete characterization of the unidentified sulfated compound present in F1 is another important aspect that deserves future investigation.
In conclusion, we believe that the present work describing the presence of these sulfated compounds in R. prolixus ovaries is timely and worthwhile, by acting as a valuable starting point for further investigation of potential roles of sulfated glycosaminoglycans in insect oogenesis.
Acknowledgements
This work was supported by Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq: PADCT and PRONEX), Fundac¸a˜o de Amparo a` Pes-quisa do Estado do Rio de Janeiro (FAPERJ) and Finan-ciadora de Estudos e Projetos (FINEP). We are grateful to Adriana A. Piquet for technical assistance.
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