34 A. Costa-Filho et al. Insect Biochemistry and Molecular Biology 31 2001 31–40
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.
35
S–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 metabolically
35
S–labeled ovaries of R. pro- lixus. The
35
S–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
35
S–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 46 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
35
S–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 ovarian
35
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–GalNAc4SO
4
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
The
35
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 sulfated proteins.
3.4. Isolation of native
35
S–sulfated compounds from fresh collected ovaries
In an attempt to obtain native
35
S–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 unidentified
35
S–sulfated compound present in Peak F1, while the
35
S–glycosaminoglycans remain entirely in the residue of the homogenization procedure
Fig. 4. The soluble
35
S–compound and the
35
S–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
35
S–compound eluted from the column as a single and sharp peak at a
slightly lower concentration of NaCl when compared with the unidentified
35
S–compound obtained by papain digestion. The soluble
35
S–compound showed a slight slower mobility on agarose gel when compared with the
papain-extracted molecule compare Fig. 4C and D. On the other hand, the results obtained analyzing the
35 A. Costa-Filho et al. Insect Biochemistry and Molecular Biology 31 2001 31–40
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 46 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
35
S–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–GlcNAc6SO
4
; Peak 2
α –
DGlcUA–1→3–GalNAc4SO
4
.
35
S–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
35
S–sulfated compound in the homo- genization-residue has decreased, as expected.
3.5. Distribution of
35
S–sulfated compounds between the ovarian tissue and the oocytes
35
S–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
In order to confirm the biochemical results on the dis- tribution of the sulfated compounds described above, we
carried out the histochemical detection of sulfated materials 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,
36 A. Costa-Filho et al. Insect Biochemistry and Molecular Biology 31 2001 31–40
Fig. 4. Anion-exchange chromatography of the native unidentified
sulfated compound extracted by homogenization of whole ovary A. The
35
S–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. The
35
S–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
35
S–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
35
S–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
35
S–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
35
S–sulfated GAG peak is cross-hatched. In C and D are shown the autoradiograms of agarose gel electrophoresis of the
35
S–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.
to this compound the metachromasia seen in this com- partment
Fig. 6D.
The metachromatic
material observed around the follicle cells as well as in the ovi-
duct Fig. 6E and C, respectively may be attributed to both sulfated glycosaminoglycans and the unidentified
sulfated compound, since both compounds could be detected
biochemically in
the fraction containing
37 A. Costa-Filho et al. Insect Biochemistry and Molecular Biology 31 2001 31–40
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
