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Biology of Reproduction 63, 913-924 (2000)
© 2000 Society for the Study of Reproduction, Inc.

Regular Article

Identification and Immunolocalization of Decorin, Versican, Perlecan, Nidogen, and Chondroitin Sulfate Proteoglycans in Bovine Small-Antral Ovarian Follicles1

Maggie E. McArthura,b, Helen F. Irving-Rodgersa, Sharon Byersb, and Raymond J. Rodgers2,a

a Department of Medicine, Flinders University of South Australia, Bedford Park, South Australia 5042, Australia b Department of Chemical Pathology, Women's and Children's Hospital, North Adelaide, South Australia 5006, Australia

ABSTRACT

Proteoglycans (PGs) consist of a core protein and attached glycosaminoglycans (GAGs) and have diverse roles in cell and tissue biology. In follicles PGs have been detected only in follicular fluid and in cultured granulosa cells, and the composition of their GAGs has been determined. To identify PGs in whole ovarian follicles, not just in follicular fluid and granulosa cells, small (1–3-mm) bovine follicles were harvested. A proportion of these was incubated with 35SO4 for 24 h to incorporate radiolabel into the GAGs. The freshly harvested and cultured follicles were sequentially extracted with 6 M urea buffer, the same buffer with 0.1% Triton X-100 and then with 0.1 M NaOH. Proteoglycans were subjected to ion-exchange and size-exclusion chromatography. The GAGs were analyzed by chemical and enzymic digestion, and on the basis of their composition, we chose a list of known PGs to measure by ELISA analyses. Versican, perlecan, decorin, but not aggrecan or biglycan, were identified. These, excluding decorin for technical reasons, as well as a basal lamina glycoprotein, nidogen/entactin, were immunolocalized. Versican was localized to the thecal layers, including externa and the interna particularly in an area adjacent to the follicular basal lamina. Perlecan and nidogen were localized to the follicular basal lamina of antral follicles, both healthy and atretic, but not to that of preantral follicles. Both were localized to subendothelial basal laminas, but the former was not readily detected in arteriole smooth muscle layers. This study has confirmed the presence of versican and perlecan, but not the latter as a component of follicular fluid, and identified decorin and nidogen in ovarian antral follicles.

follicle, follicular development

INTRODUCTION

Development of mammalian ovarian follicles and oocytes is a complex process involving tissue growth and remodeling, fluid accumulation, and cell replication, specialization, and differentiation. Proteoglycans (PGs) are ubiquitous molecules of extracellular matrices that have been implicated in these processes in a variety of other tissues [1]. Proteoglycans consist of glycosaminoglycans (GAGs) covalently attached to a protein core. Glycosaminoglycans consist of chains of repeating disaccharides that vary in composition thus forming different GAG chains. Proteoglycans are therefore a diverse range of molecules with diverse functions, documented to be involved in cell growth and differentiation, water homeostasis, and the regulation of growth factors.

Glycosaminoglycans have been identified in the ovarian follicular fluid of pigs [2–4], cows, [5–9], humans [10–12], and rats [13, 14]. The predominant GAGs in bovine and porcine follicular fluid are dermatan sulfate (DS) and chondroitin sulfate (CS) [2, 6]. The CS/DS-containing GAGs were shown to be attached to a protein core in bovine follicles by Grimek and Ax [9], while the GAG heparan sulfate (HS) was observed not to be bound to a protein core. The concentration of GAGs in bovine follicular fluid varied with size and health of the developing follicles [8, 9]. Chondroitin sulfate concentrations were higher in the follicular fluid of small-antral follicles as compared with large antral follicles [9]. The concentration of CS was also reported to vary with the health of the ovarian follicle but only when follicular atresia was assessed biochemically, not histologically [8].

The size of follicular fluid PGs was estimated to range from 7.5 x 105 to 2 x 106 [2, 9]. Molecules of this size are too large to traverse the basal lamina readily, thus they must be produced within the follicle by the granulosa cells [2]. Bovine granulosa cells in anchorage-independent culture produced an extracellular matrix rich in PGs, as determined by ruthenium red staining [15]. Cultured rat granulosa cells also produced PGs; determined by the incorporation of radiolabeled sulfate. These PGs included three hydrodynamic sizes of DS-containing PGs and two different hydrodynamic sizes of HS-containing PGs [16–19]. These DS- and HS-containing PGs were also found to be either intracellular, cell surface associated, or bound within the cell membrane. Production of CS/DS PGs by granulosa cells has been shown to increase in response to FSH in vitro [4, 7, 20]. Recently some of the PGs in human follicular fluid were identified as a versican-like PG and perlecan [12].

In addition to these PGs, other PGs could be present in follicular fluid, and no studies of PGs in the other follicular compartments have been conducted. We therefore analyzed, identified, and immunolocalized a number of PGs in bovine whole antral follicles. Small follicles were used as they are mostly healthy and at an early stage of antrum expansion.

MATERIALS AND METHODS

Tissues

All bovine ovaries were collected at a local abattoir, within 20 min of slaughter, from cyclic cows visually assessed not to be pregnant. The ovaries were immediately placed into Hepes-buffered Earle's balanced-salt solution without calcium and magnesium, containing 10 mM N-ethylmalemide (NEM), 5 mM benzamidine, 0.5 mM PMSF, 0.1 M {delta}-amino-caproic acid, and 0.01 M EDTA, on ice for return to the laboratory. Antral follicles of 1–3 mm diameter were dissected from the whole ovary. Most of these were weighed, snap frozen, and stored at -20°C for analyses of PGs. For analyses of PGs, 18 separate batches of 1- to 3-mm follicles from 18 ovaries were isolated. These were subsequently pooled and weighed 4.633 g. Three antral follicles from each batch were randomly chosen and fixed for histological assessment of follicular health. Other follicles were taken for culture as below and, following culture, were pooled with the unicubated batches prior to isolation of PGs.

Histological Assessment of Follicular Health

Dissected follicles were fixed by immersion in 2.5% glutaraldehyde in 0.1 M phosphate buffer for 24 h at 4°C. Subsequently, specimens were washed in 5% sucrose in 0.1 M phosphate buffer, postfixed in aqueous 1% osmium tetroxide for 60 min, dehydrated using increasing concentrations of acetone, then embedded in epoxy resin (49% dodecenyl succinic anhydride, 39.2% Araldite, 9.8% Procure 812, 2% 2,4,8-tridimethylaminoethyl phenol), and cured at 60°C. Sections of 1 µm were cut, stained with 1% methylene blue in 1% sodium tetraborate, and examined by light microscopy. Follicles were assessed as atretic if pyknotic nuclei were present in the membrana granulosa.

Radiolabeling of Follicular PGs

In order to increase the sensitivity of detection the GAGs they were radiolabeled during culture of follicles. To do this, ovaries were collected as described above and immediately placed into Hepes-buffered Dulbecco's-modified Eagle's medium (DMEM) : Ham's F12 medium (50:50) containing 100 µg/ml penicillin, 100 µg/ml streptomycin, and 0.25 µg/ml fungizone on ice. Antral follicles of 1–3 mm diameter were dissected from the ovaries, then washed in the same medium but without Hepes. Three batches of follicles were collected from three ovaries, weighing 0.78 g collectively. Dissected follicles were cultured in 35-mm dishes in DMEM/Ham's F12 medium containing 1% fetal bovine serum, antibiotics, and 20 µCi/ml 35SO4 (562.5 Ci/mM) for 24 h in a humidified atmosphere of 5% CO2 in air at 39°C. Follicles were then harvested, snap-frozen on dry ice, and stored at -20°C for further analyses.

PG Extraction

The batches of 35S-labeled follicles were combined with the batches of unlabeled follicles. These pooled follicles (5.43 g) were minced with a razor blade and then extracted with five volumes of 6 M urea solution containing 0.05 M sodium acetate, 0.1 M {delta}-amino-caproic acid, 0.1 M disodium salt EDTA, 5 mM benzamidine, 0.5 mM PMSF, and 10 mM NEM, pH 5.0, by continuously rotating the solution at 4°C for 48 h. Following centrifugation (6000 x g for 15 min), the supernatant was retained for analyses and stored at -20°C. This will be referred to as the urea extract.

The pellet was then placed into five volumes of the urea extraction buffer described above but also containing 0.1% Triton X-100 and homogenized using a Dounce teflon/glass homogenizer for 10 min and rotated at 4°C for 48 h. Following centrifugation (6000 x g for 15 min), the supernatant was retained for analyses and stored at -20°C. This will be referred to as the urea/Triton X-100 extract.

The pellet was placed into 10 ml of 0.1 M sodium hydroxide to solubilize the remaining tissue components by rotation at 4°C for 48 h. The extract was then centrifuged (6000 x g for 15 min), and both the pellet and the supernatant were retained for analyses at -20°C. The supernatant will be referred to as the sodium hydroxide extract.

Glycosaminoglycan and Protein Measurements

After each step of chromatographic analyses the GAG content of samples was measured using either the carbazole method published by Blumenkrantz and Asboe-Hansen [21], or the dye-binding assay published by Farndale et al. [22]. Protein analyses were carried out using the bicinchoninic acid method of Smith et al. [23]. Radioactivity of samples was measured by scintillation counting.

Ion-Exchange Chromatography

The three sequential follicle extracts were each chromatographed using ion-exchange chromatography. The pH of the sodium hydroxide extract was first adjusted to pH 5.0 with hydrochloric acid. Each extract was rotated for 1 h with 2 ml of DEAE Sephacel beads (Pharmacia, Uppsala, Sweden) at 4°C. The DEAE Sephacel columns were then washed with 6 M urea, 0.05 M sodium acetate, 0.1 M {delta}-amino-caproic acid, 0.1 M disodium salt EDTA, pH 5.0, to remove tissue components unbound to the column beads. The PGs were then sequentially eluted from the columns in urea buffer above containing 0.15 M NaCl, followed by urea buffer containing 2.0 M NaCl. All eluted fractions were assayed for GAGs using the carbazole method. Glycosaminoglycan containing-fractions from the urea and urea/Triton X-100 extracts were pooled together due to the low amount of GAG present in the extracts. This will now be referred to as the urea-urea/Triton X-100 extract. The GAG-containing fractions from the sodium hydroxide extract were also pooled. All pooled fractions were retained for analyses and stored at -20°C.

Size-Exclusion Chromatography

Pooled GAG-containing fractions from above were dialyzed against 0.1 M Tris acetate buffer and then against deionized water before lyophilization and resuspension in 500 µl of column buffer containing 2 M guanidine, 0.1 M sodium acetate, 0.05 M Tris, pH 7.5. The resuspended urea-urea/Triton X-100 extract was chromatographed on a 90-cm sepharose CL2B (Pharmacia) column at a flow rate of 4 ml/h. Forty milliliters of column buffer was eluted (80 fractions). Fractions were assayed for GAG using the dye-binding method, and aliquots were also taken for liquid scintillation counting. Aliquots (200 µl) of each fraction were dialyzed against 0.1 M Tris acetate buffer, pH 7.0, followed by deionized water, and then lyophilized. The PG composition of the column fractions from the urea-urea/Triton X-100 extracts was determined by ELISA using a panel of antibodies specific to various PGs as described below.

The resuspended sodium hydroxide extract was similarly chromatographed but on a 90-cm sepharose CL4B column. The fractions containing GAGs were pooled, dialyzed, lyophilized, and resuspended in 150 µl H2O, and the GAG composition determined by nitrous acid cleavage and chondroitinase digestions as described below.

Glycosaminoglycan Characterization of Sodium Hydroxide Extract

Nitrous acid cleavage An aliquot (20 µl) was mixed with nitrous acid to cleave HS side chains as described by Hopwood [24]. The sample was incubated for 1 h at room temperature and then neutralized with sodium carbonate. Samples (20 µl mixed with 480 µl column buffer as above) were rechromatographed on a sepharose CL4B column as described above.

Chondroitinase digestion An aliquot (20 µl) was treated with chondroitinase ACII lyase (EC 4.2.2.5; ICN Immunobiologicals, Costa Mesa, CA) to digest CS side chains. The digestion was carried out in 250 µl of 0.08 M Tris acetate buffer, pH 6.0, containing 0.02 units of enzyme for 4 h at 37°C [25]. An aliquot was also digested with chondroitinase ABC lyase (EC 4.2.2.4; ICN Immunobiologicals) to digest both CS and DS side chains. The digestion was carried out in 240 µl of 0.1 M Tris acetate buffer, pH 8.0, using 0.02 units of enzyme for 4 h at 37°C [25]. Samples were rechromatographed on a sepharose CL4B column as described above.

Primary Antibodies and Purified PGs

Primary antibodies directed against epitopes present on 4-sulfated CS/DS (murine monoclonal antibody 2B6, IgG purified from ascites fluid, recognizes a disaccharide containing a nonreducing 4,5 unsaturated hexuronate adjacent to a 4-sulfated N-acetylgalactosamine that is produced by chondroitinase digestion of native CS or DS chains), 6-sulfated CS/DS (murine monoclonal antibody 3B3, IgM purified from ascites fluid, recognizes a disaccharide containing a nonreducing unsaturated hexuronate adjacent to a 6-sulfated N-acetylgalactosamine which is produced by chondroitinase digestion, or on nondigestion it recognizes an epitope on native CS chains containing a nonreducing unsaturated or saturated hexuronate adjacent to a 6-sulfated N-acetylgalactosamine) [26, 27], and aggrecan (murine monoclonal antibody 1C6, raised against reduced and alkylated rat chondrosarcoma PGs) [27] were kindly donated by Bruce Caterson, Department of Surgery, University of North Carolina at Chapel Hill, NC. Antibodies recognizing the bovine decorin (rabbit serum, antibody LF94, raised against a synthetic peptide) and the bovine biglycan (rabbit serum, antibody LF96, raised against a synthetic peptide) were kindly donated by Larry Fisher, Bone Research Branch, National Institute of Dental Research, National Institutes of Health, Bethseda, MD [28–30]. The antibody recognizing the bovine versican, was affinity-purified IgG from a rabbit immunized with a histidine-tagged recombinantly expressed and purified fragment (amino acid numbers 1340–1613) of bovine versican called GAG-ß [31]. It was kindly donated by Dieter Zimmermann and Maria Teresa Dours-Zimmermann, Department of Pathology, University of Zurich, Schmelzbergstrasse, Zurich, Switzerland. The antibody recognizing the HS-containing PG perlecan (murine monoclonal antibody A76, raised against bovine corneal endothelial cell extracellular matrix) was kindly donated by Anne Underwood, CSIRO Molecular Science, North Ryde, NSW, Australia [32]. The antibodies recognizing nidogen/entactin, called 913 and 914, were rabbit sera raised against purified nidogen isolated from mouse EHS tumor [33] and were kindly donated by Dr. Marie Dziadek, Department of Anatomy, University of Melbourne. Purified decorin was isolated from bovine articular cartilage in house.

Enzyme-Linked Immunosorbent Assay Analyses of Urea-Urea/Triton X-100 Extract

Samples (10 µl) to be tested were diluted in 140 µl of PBS (10 mM) and incubated (1 h at 37°C) in 96-well plates to allow attachment to wells. The wells were then washed in PBS and incubated with 150 µl of 0.01 M PBS containing 1% BSA (1 h at 37°C). Primary antibody at the appropriate dilution (1/5000 for 2B2 and 3B3, and 1/1000 for 1C3, LF94, LF96, GAG-ß, and A76) in 150 µl of the same buffer was added to each well and incubated (1 h at 37°C). The secondary antibodies were horseradish peroxidase-conjugated sheep anti-mouse IgG (Silinus Laboratories, Vic, Australia) for monoclonal primary antibodies, and horseradish-peroxidase-conjugated sheep-anti-rabbit IgG (Silinus Laboratories, Vic, Australia) for polyclonal antibodies were applied at a dilution of 1/500 in same buffer and incubated (1 h at 37°C). The peroxidase activity was measured as a color reaction using 100 µl of 2,2'-azino-di-(3-ethylbenzthiazoline-6-sulfonic acid) as substrate with 10% hydrogen peroxide, prepared as described in the BioRad horseradish peroxidase substrate kit (BioRad, Hercules, CA). The optical density was read at 415 nm.

Samples to be tested for the presence of aggrecan were, prior to attachment to the wells, first reduced by treatment with 10 mM DL-dithiothreitol (100°C for 10 min) and then alkylated in 20 mM iodoacetic acid (in dark for 2 h) in order to expose the antigen. Samples to be incubated with the 2B6 or 3B3 antibodies required prior digestion with chondroitinase ABC lyase to expose the antigen. Chondroitinase ABC lyase was prepared at 0.5 units per 10 ml, 0.1 M Tris acetate buffer, pH 8.0, containing 1% BSA. The digestion occurred in the 96-well plates in which the samples had previously been incubated to allow attachment, by adding 150 µl to each well and incubating at (37°C for 1 h).

Immunohistochemistry

The antibodies used for immunohistochemistry were LF94 recognizing decorin, A76 recognizing perlecan, 2B6 recognizing chondroitin/dermatan-4-sulfate, GAG-ß recognizing versican, and 913 recognizing nidogen/entactin are described above. Formalin-fixed and paraffin-embedded human rib sections were used as the positive control tissue for 2B6 antibody. Paraffin-embedded sections were dewaxed and rehydrated by sequential incubations in xylene, absolute ethanol, 90% ethanol, 70% ethanol, and water. Frozen kidney sections (10 µm) were used as positive control tissue for LF94 and A76. Sections of frozen tissues used for decorin, chondroitin/dermatan-4-sulfate, versican, and nidogen localization were fixed in 4% paraformaldehyde solution for 20 min (n = 6 ovaries). Frozen sections to be used for perlecan localization were fixed in periodate-lysine-paraformaldehyde fixative for 20 min (n = 4 ovaries).

After fixing or rehydrating, sections were washed in hypertonic PBS containing 0.274 M sodium chloride, 5.37 mM potassium chloride, 10 mM sodium phosphate, pH 7.2. Sections to be incubated with 2B6 or LF94 required predigestion with chondroitinase ABC lyase to expose the antigen. Fifty microliters of chondroitinase ABC lyase at a concentration of 0.05 units/ml in 0.1 M Tris acetate buffer, pH 8.0, was applied to each slide for 1 h at 37°C. The slides were washed in hypertonic PBS. All slides were blocked in hypertonic PBS containing 10% normal donkey serum (NDS) for 30 min at room temperature and then incubated with primary antibody diluted in hypertonic PBS containing 10% NDS (antibody dilutions for A76 was 10 µg IgG/ml or 1/200, LF94 1/200, 2B6 1/1000, GAG-ß 1/1000 and for 913 1/200, negative controls normal mouse serum and normal rabbit serum 1/100) for 1 h. Sections were then rinsed and incubated in a 1/25 dilution of biotin conjugated anti-mouse or anti-rabbit IgG (biotin-SP-conjugated AffiniPure F (ab')2 fragment donkey anti-mouse or rabbit IgG (H&L); Jackson ImmunoResearch Laboratories, West Grove, PA) for 30 min. Sections were then rinsed and incubated in avidin-biotin complex (VectorStain ABC Kit PK 4001 from Vector Laboratories Inc., Burlingame, CA) diluted 1/100 in hypertonic PBS for 15 min. Sections were then rinsed and then peroxidase detected using substrate diaminobenzidine from the DAKO Liquid DAB+ Substrate-Chromagen System (DAKO Corporation, Carpinteria, CA; catalog no. K3467). The sections were then rinsed and mounted in buffered glycerol and examined by light microscopy.

RESULTS

Ovarian Follicles

A total of 21 batches of 1–3-mm follicles from 21 ovaries were isolated, weighing a total of 5.43 g. Histological assessment was carried out on a random sample size of about 10% of the isolated follicles and revealed that 85% of follicles were healthy, while 15% were in early atresia, as determined by pyknosis of the granulosa cell layer. There were no regressed follicles present. The freshly isolated follicles and the 35S-labeled follicles were pooled for further analyses.

Extraction of PGs

Three sequential extractions were carried out on the pooled follicles to separate the PGs into groups based on their cellular location. The initial urea extraction was designed to remove all soluble PGs, the urea plus Triton X-100 extraction to extract membrane-associated PGs, and the sodium hydroxide extraction to solubilize the remaining tissue components. Thus, all constituents of the follicles were examined. Protein yields were 172 mg in the urea extract, 81 mg in the urea/Triton X-100 extract, and 85.3 mg in the sodium hydroxide extract. Glycosaminoglycan analyses of these samples were not possible due to high protein levels interfering with the GAG assay.

Ion-Exchange Chromatography

The urea, urea/Triton X-100, and sodium hydroxide extracts were chromatographed on a DEAE Sephacel column to separate the PGs from the majority of the proteins present. The majority of GAGs were recovered from the column with 2.0 M NaCl buffer (Fig. 1). The urea extract elution profile (Fig. 1A) revealed that 94.6% of protein and 98.7% of radiolabeled components were present in the flow through and urea buffer washes, while 85% (110.29 µg) of the GAG was eluted with 2.0 M NaCl. Thus, the majority of protein was separated from the PGs present. This pattern was also observed in the urea/Triton X-100 extract (Fig. 1B) in which 95.9% of protein and 88.7% of radiolabeled components were present in the flow through and the urea wash, separated from 80% (34.59 µg) of GAG that eluted with 2.0 M NaCl. The sodium hydroxide extract (Fig. 1C) showed 93.8% of protein and 42% of radiolabeled components in the flow through and urea wash. Eighty-eight percent (29.5 µg) of GAG eluted with 2.0 M NaCl as did 44% of radiolabeled components. Thus, in contrast to the previous two extracts, the GAGs in the sodium hydroxide extraction were radiolabeled.



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  		FIG. 1. Anion-exchange chromatography on a DEAE cellulose column (2 ml) of each of the sequential tissue extracts made from small-antral bovine follicles: A) urea extract, B) urea/Triton X-100 extract, and C) sodium hydroxide extract. The column was developed with the urea extraction buffer (6 M urea, 0.05 M sodium acetate, 0.1 M {delta}-amino-caproic acid, 0.1 M disodium salt EDTA, pH 5.0; urea wash), followed by 0.15 M NaCl, and then 2.0 M NaCl in the urea extraction buffer. The eluted fractions were analyzed for their concentrations of protein ({diamondsuit}), GAGs (•), and 35S ({triangleup})

The GAG-containing fractions for each extract that eluted with 2 M NaCl were pooled for further analyses. As the urea/Triton X-100 extract contained only 34.6 µg of uronic acid (fractions 11–12, Fig. 1B) these were pooled with the urea extract (fractions 11–12, Fig. 1A) to allow further analyses. This will now be referred to as the urea-urea/Triton X-100 extract. The sodium hydroxide extract contained only 29.5 µg of uronic acid, but as the PGs were affected by the sodium hydroxide treatment and because the GAGs in this extract were radiolabeled, these fractions (fractions 11–12, Fig. 1C) were maintained as a separate pool for further analyses.

Size-Exclusion Chromatography

The GAG-containing fractions of the urea-urea/Triton X-100 extracts (fractions 10–13 in Fig. 1A and fractions 11–12 in Fig. 1B) were combined and chromatographed on a sepharose CL2B column (Fig. 2A). A small peak of a large PG eluted with a Kav range 0.24 to 0.57 (Fig. 2A, bar 1). A large polydisperse peak of GAG eluted with a Kav range of 0.57 to 0.81 (Fig. 2A, bar 2) and had a shoulder ranging up to 1.0 (Fig. 2A, bar 3). All column fractions were retained for ELISA analyses.



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  		FIG. 2. Size-exclusion chromatography of PGs isolated by anion-exchange chromatography of three sequential extracts of small-antral bovine follicles. A) The urea and urea/Triton X-100 isolates were combined and then chromatographed on 90-cm size-exclusion sepharose CL2B column (elution buffer of 2 M guanidine, 0.1 M sodium acetate, 0.05 M Tris, pH 7.5); 1, 2, 3 represent three peaks of PGs. B) The sodium hydroxide isolates were chromatographed on 90-cm size-exclusion sepharose CL4B column; 1 represents a peak of PG. The eluted fractions were analyzed for their concentrations of protein ({diamondsuit}), GAGs (•), and 35S ({triangleup}). No 35S was detected in A

The GAG-containing fractions of the sodium hydroxide extract (fractions 11–12, Fig. 1C) were combined and chromatographed on a sepharose CL4B column (Fig. 2B). A single polydisperse peak of GAG, corresponding to a peak of radioactivity eluted with a Kav range of 0.5–0.9 (Fig. 2B, bar 1). Fractions from this peak were pooled for GAG identification by enzyme digestion.

Characterization of GAGs

Aliquots of the sodium hydroxide extract were digested with GAG-degrading enzymes and rechromatographed on the sepharose CL4B size-exclusion column. The degree of GAG digestion was assessed by calculating the percentage of radiolabeled PGs that had shifted from the undigested PG peak (elution volume 17–27 ml, Fig. 3A) after enzyme degradation or nitrous acid cleavage. Of the PGs present 20.3% were susceptible to nitrous acid cleavage, indicating the presence of HS (Fig. 3B), 38.7% were susceptible to chondroitinase ACII digestion (Fig. 3C), and 70% were susceptible to chondroitinase ABC lyase digestion (Fig. 3D). Thus, the PG(s) present in the sodium hydroxide extract contained a majority of CS, followed by DS, followed by a small amount of HS.



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  		FIG. 3. Glycosaminoglycan characterization of PGs isolated from the sodium hydroxide extract of small-antral bovine follicles by ion-exchange chromatography. These PGs were either A) untreated, B) treated with nitrous acid to cleave HS, C) digested with chondroitinase ACII lyase to remove CS, or D) digested with chondroitinase ABC lyase to remove CS and DS. These digests were each separately chromatographed on a 90-cm size-exclusion sepharose CL4B column (elution buffer of 2 M guanidine, 0.1 M sodium acetate, 0.05 M Tris, pH 7.5), and the 35S in each eluted fraction was measured

Identification of GAGs and PGs

The presence of different GAGs and PGs in individual column fractions from the CL2B chromatograph of the urea/Triton X-100 extract was determined by testing column fractions for reactivity to antibodies against defined GAG and PG epitopes. Positive controls of purified PG preparations and PGs containing the appropriate GAGs were included.

The antibody 2B6 that recognizes 4-sulfated CS/DS GAG identified three peaks of reactivity (Fig. 4A) that corresponded to the GAG profile observed earlier (Fig. 2A). This indicates the presence of more than one CS/DS-containing PG in the urea-urea/Triton X-100 extract. Reactivity to the antibody 3B3 was also tested; however, no reactivity was observed, indicating that there was no or little CS/DS-6-sulfated GAG in this extract.



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  		FIG. 4. Characterization of PGs isolated by ion-exchange chromatography of the urea-urea/Triton X-100 extract of small-antral bovine follicles. These PGs were chromatographed on a size-exclusion 90-cm sepharose CL2B column and the eluted fractions were analyzed by ELISA for A) chondroitin/dermatan-4-sulfate, B) versican, C) decorin, and D) perlecan

Reactivity to the antibody GAG-ß that recognizes the CS PG versican was also tested (Fig. 4B). A polydisperse peak of reactivity was observed that overlapped the eluted second peak of 2B6 reactivity. Antibodies to components of the large aggregating cartilage PG aggrecan and to the PG biglycan were also tested; however, no reactivity was observed (data not shown). There was no reactivity observed in the sodium hydroxide extract to any of the antibodies tested. This may have been due to sodium hydroxide extract containing only a small amount of PG or to a reduction in the ability of any PGs to bind to the ELISA tubes following the sodium hydroxide treatment.

The antibody LF94 recognizes the core protein of decorin, which is a 4-sulfated CS/DS PG. One peak of reactivity to LF94 was observed (Fig. 4C) that corresponded to the third eluted peak of CS/DS-containing PGs observed using the 2B6 antibody (Fig. 4A). Thus, it appears that decorin is present in the follicle extract. To confirm that this peak corresponded to decorin, purified decorin isolated from bovine articular cartilage was also chromatographed on the CL2B column. This purified decorin preparation eluted in the same position as both the decorin and 4-sulfated CS/DS antibody reactive peaks observed in the extract (results not shown).

Reactivity to the antibody A76 that recognizes the HS PG perlecan was also tested (Fig. 4D). One peak of reactivity was observed indicating the presence of perlecan. This peak coincided with the later fractions that eluted in the second peak of 2B6 reactivity.

These results indicate the presence of a large amount of 4-sulfated CS/DS reactivity in the urea-urea/Triton X-100 extract. One portion of this CS/DS reactivity has been identified as the DS SLRP decorin in peak 3 (Fig. 4), and versican was also identified in peak 2. The modular HS PG perlecan was also observed in the extract. The remainder of CS/DS reactivity was unaccounted for, indicating the presence of larger unidentified PGs in peak 1.

Immunolocalization of PGs

In antral follicles CS/DS-4-sulfate immunoreactivity was observed extracellularly in the theca interna of healthy (Fig. 5A), atretic, and regressing antral follicles, with highest concentration adjacent to the follicular basal lamina (n = 6 ovaries examined). The follicular basal lamina may have been positively stained, but Call-Exner bodies (result not shown) were not stained, suggesting that the CS/DS-4-sulfate may not be a component of the follicular basal lamina. Weaker staining was observed in the membrana granulosa, and the staining was extracellular. Not all follicles (Fig. 5B) had the same staining pattern; in some the staining was weaker adjacent to the basal lamina. In addition CS/D-4-sulfate immunoreactivity was localized to filamentous material within the theca externa layers (Fig. 5B) and in the connective tissue septa surrounding large blood vessels (Fig. 5D). No staining was associated with primordial or preantral follicles (Fig. 5C). Negative control sections were those in which normal mouse serum was substituted for primary antisera (Fig. 6, A and B) or where primary antisera was employed but without prior chondroitinase digestion required to expose the antigen (not shown). No staining was observed in these controls.



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  		FIG. 5. Immunolocalization of 4-sulfated CS/DS in the bovine ovary. In many antral follicles (A), but not all (B), staining was strongest in the theca interna (TI) adjacent to the basal lamina (arrows), and weaker staining was present in the membrana granulosa (G). Staining was associated with fibrillar structures within ovarian stroma tissue or in the theca externa (TE). Staining was absent from primordial (arrows) and preantral follicles (C). 4-Sulfated CS/DS was immunolocalized to the connective tissue sheaths surrounding bloods vessels (D). Scale bars = 10 µm in A and B, 20 µm in C, and 50 µm in D. V, Blood vessel



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  		FIG. 6. A, B) Control sections of bovine antral follicles in which the primary antisera have been replaced by normal mouse serum show no positive staining. C, D) Control sections of bovine follicles in which the primary antisera have been replaced by normal rabbit serum show no positive staining. Sections B and D were pretreated with chondroitinase ABC lyase. A = 170 x 240-µm follicle; B = 1-mm follicle; C = 90-µm preantral follicle; D = 275 x 375-µm antral follicle. Scale bars = 20 µm in A, C, and D, and 50 µm in B

Primordial and preantral follicles showed little or undetectable levels of immunoreactivity for versican (n = 6 ovaries examined) (Fig. 7A). Immunoreactivity was observed extracellularly in the theca interna of healthy antral follicles (Fig. 7B), in high concentration at or near the follicular basal lamina. Additional staining was observed extracellularly in the more antrally situated granulosa cells. No staining was observed in Call-Exner bodies (result not shown). Strong immunoreactivity was seen in atretic (Fig. 7C) and regressed (Fig. 7D) follicles in the area occupied by the theca interna. Normal rabbit serum was used in place of primary antisera in negative control sections (Fig. 6, C and D).



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  		FIG. 7. No positive staining for versican was observed in association with primordial or preantral follicles (A, arrows). Staining was concentrated at the theca side but toward the follicular basal lamina (arrows in B and C) in healthy follicles (B) but more so in severely atretic (C) and regressed (D) follicles. Scale bars = 20 µm in A, 50 µm in B and C, and 100 µm in D

In all healthy antral follicles examined perlecan was immunolocalized (n = 4 ovaries) to the follicular basal lamina separating the membrana granulosa from the surrounding thecal layers (Fig. 8, A and B). Call-Exner bodies (Fig. 8A) present in some early or small-antral follicles were positively stained. Perlecan was also detected in the follicular basal lamina of atretic antral and regressed follicles, indicating that perlecan is not lost during follicular regression. Staining was weak or undetectable in preantral follicles (results not shown). No staining was observed in the membrana granulosa or in the thecal cell layers apart from that associated with blood vessels in the theca. Strong perlecan immunoreactivity was seen in the subendothelial basal laminas of capillaries, arterioles, and larger blood venules in both the theca and ovarian stroma. Less intense staining was localized to the basal laminas associated with ovarian arteriolar smooth muscle cells. The negative control used was normal mouse serum. There was no staining observed within the ovary (Fig. 6B) when normal mouse serum was substituted for the primary antibody.



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  		FIG. 8. Immunolocalization of perlecan (A and B) and nidogen (C and D). Perlecan localized to the follicular basal lamina (A and B, arrows), Call-Exner bodies (arrowheads in A), and subendothelial basal laminas (arrowheads in B). Nidogen was absent from the basal lamina (arrow or arrowhead in C) in primordial and preantral follicles but was a component of the follicular basal lamina of antral follicles (D), Call-Exner bodies (arrowheads in D), and blood vessels (arrows in D) within the ovary. Panel A shows a 375 x 500-µm follicle, B shows a 125 x 250-µm follicle, C shows primordial (arrow, 18 x 34-µm) and preantral (arrowhead, 30 x 55-µm) follicles, and D shows a 250 x 300-µm follicle. Scale bars = 20 µm in A, B, and D, and 10 µm in C

Immunoreactivity to nidogen (n = 6 ovaries) was confined to basal laminas of structures within ovary. Little or undetectable staining was observed in primordial and small-antral follicles (Fig. 8C); however, within antral follicles nidogen was immunolocalized to follicular basal lamina of healthy, atretic, and regressed follicles. Call-Exner bodies present in early and small-antral follicles also stained positively (Fig. 8D). Nidogen was also immunolocalized to the subendothelial basal laminas of the ovarian vasculature (capillaries, venules, and arterioles) and the basal lamina associated with arteriolar smooth muscle cells (Fig. 8D). No staining was observed when normal rabbit serum was substituted for the primary antisera in control sections (Fig. 6, C and D).

No specific staining was observed in the ovary sections incubated with the antibody LF94 to decorin (results not shown). In spite of this antibody reacting with solubilized decorin in the ELISA assays it is suspected that decorin in tissue is in a different confirmation and thus unable to be detected by this particular antibody.

DISCUSSION

This study identified PGs in small bovine ovarian follicles, not just follicular fluid. Radiolabeled sulfate was incorporated into the GAG chains of PGs synthesized during culture of follicles. Radiolabeled PGs were present only in the sodium hydroxide extract, when clearly there was a high yield of GAGs in the other extracts, implying that these GAGs were not from the same cellular pool. Thus we analyzed both PGs with unlabeled GAGs by quantitating GAGs directly and by ELISA assay, and PGs with labeled GAGs by enzymic and chemical digestion of the GAG chains from the PGs. Following column chromatography versican, decorin, and perlecan were identified; aggrecan and biglycan were not detected. Versican and perlecan, and a basal lamina component, nidogen/entactin were then immunolocalized in ovarian follicles.

The GAGs newly synthesized by the follicles in culture in the current study had both CS and DS and less HS. Glycosaminoglycans with DS and CS have previously been documented in the follicular fluid of bovine, porcine, and human ovarian follicles [2, 5–9, 11, 12]. Heparin sulfate has also been observed in rat ovarian slices [13] and in human follicular fluid [10]. The CS/DS GAGs were mostly 4-sulfated. This is also the case in bovine follicular fluid GAGs [6], while porcine follicular fluid contains unsulfated, 4-sulfated, and 6-sulfated CS/DS GAG chains [2], and human follicular fluid contains both unsulfated and 6-sulfated GAGs. Thus there is clearly species variation in the level and type of sulfation of the GAGs in follicles.

The PGs containing CS/DS identified in the current study were a diverse group of PGs. Three peaks of immunoreactivity for 4-sulfated CS/DS were identified by size-exclusion chromatography, corresponding to three peaks of GAGs as measured by uronic acid analyses. This indicated the presence of at least three species of PGs within the follicles that were relatively rich in CS/DS. The first peak (Kav 0.24–0.57) indicated that the PGs were of similar size to a CS/DS PG reported in the follicular fluid of bovine follicles by Grimek et al. [2]. Yanagishita et al. [2] also characterized a PG of similar size (Kav 0.26) in porcine follicular fluid. This PG was found to have a core protein of approximately 400 000, with an average of 20 DS chains attached. Thus, it appears that some PGs present in both bovine and porcine follicles are of similar sizes. This peak overlapped the peak of immunoreativity of versican, and whether this peak contains only versican remains to be determined.

The second, but broad peak of CS/DS reactivity eluted from a sepharose CL2B column with a Kav range of 0.57–0.81. This again confirms the observations of Grimek et al. [6], who documented the majority of GAGs of bovine follicular fluid eluted from a sepharose CL2B column with a Kav of 0.65. We were able to show that peak 2 also coincided with immunoreactivity to the antibody GAGß1, specific to the core proteins of bovine versican. Versican contains CS, but whether there are further CS-containing GAGs in the second broad peak has still to be determined. Cultured rat granulosa cells have also been observed to produce an HS PG in this size range, of Kav 0.62 [16, 17]. Perlecan a basal lamina HS PG, produced by human colon carcinoma cells [34] has been characterized by sepharose CL2B chromatography to have a Kav of 0.57 [35]. Thus on this basis of reactivity to antibody A76 we were able to identify perlecan in extracts from follicles, eluting with the same elution profile as human perlecan [35]. This overlapped the second peak of GAGs identified.

The third peak of 2B6 reactivity had a Kav range of 0.81–1.0 on the CL2B column. A PG of this size has been observed in both bovine [6, 8] and porcine [2] follicular fluid; however, it was not identified by either investigators. The results demonstrate that this peak of 2B6 reactivity corresponds to a peak of reactivity to the antibody LF94, specific to the core protein of the CS/DS-containing PG, decorin. The presence of decorin was further confirmed by chromatographing a purified preparation of bovine decorin on the sepharose CL2B column, showing it to elute in the same position as the decorin immunoreactivity isolated from bovine follicles.

On identifying these PGs they were then immunolocalized in the ovary. The CS/DS sulfate was localized to the follicular basal lamina and the thecal and granulosa cell layers within the antral ovarian follicle. This localization pattern, of course, represents the presence of different PGs containing CS/DS GAGs. Versican is a large CS-containing PG secreted by fibroblasts. It has some domains highly homologous to aggrecan, and it has a hyaluronan-binding domain. Fibroblasts are present in the thecal layers but not in the epithelioid membrana granulosa. In agreement, versican was observed extracellularly in the theca interna of healthy antral follicles and at high concentrations at or near the follicular basal lamina. No staining was observed in Call-Exner bodies of the membrana granulosa, suggesting that if the versican is actually associated with the follicular basal lamina, it is not derived from the granulosa cells, as other components appear to be [36]. Strong immunoreactivity was seen in atretic and even regressed follicles.

Decorin was another CS/DS-containing PG identified here, but it could not be immunolocalized, presumably because the antibody would not recognize decorin by immunohistochemistry methods. However, decorin is a ubiquitous PG that is found associated with type I collagen fibrils throughout the body [37]. Decorin is known to regulate collagen fibril formation and thus is important in the development of the structure of the extracellular matrix [38]. The follicular decorin could be associated with type I collagen that has been shown to be present within the thecal layers of the bovine follicle [39]. In addition, decorin may also influence folliculogenesis by interacting with, and perhaps sequestering, growth factors such as transforming growth factor (TGF)ß [40, 41]. Transforming growth factor-ß has been identified in the theca of a number of species and effects on granulosa cells demonstrated [42]. Thus, decorin may not only be important in follicles for collagen fibril formation, it may also be involved in regulating the activity of TGFß.

Perlecan is an HS-containing PG and was localized to the follicular basal lamina of antral ovarian follicles, both healthy and atretic follicles. This indicates that perlecan is not lost from the follicular basal lamina during atresia and regression. It has previously been observed that the follicular basal lamina contains an unidentified HS-containing PG [43], and the results of this study suggest that this PG may be perlecan. Immunostaining of perlecan in primordial or preantral ovarian follicles was weak or undetectable in the follicular basal lamina. Thus in the follicular basal lamina perlecan, like subunits of laminin [44] and collagen IV [45], appears to be developmentally regulated. Generally the main function of perlecan in the basal lamina is thought to be structural [46]. Perlecan has been shown to interact with other components of the basal lamina, including laminin, [47], type IV collagen [48], and fibronectin [49]. It is likely that perlecan thus stabilizes the follicular basal lamina during folliculogenesis.

The presence of perlecan in the follicular basal lamina may also have an important role in formation of follicular fluid. It has been proposed that the basal lamina acts as a selective filter of serum during fluid accumulation [50]. Perlecan has been implicated in the filtration of serum in the glomerulus of the kidney [51], due to the large anionic charge of the HS GAG chains. Thus, perlecan expression at the time of antrum formation may be a key event in follicular fluid formation.

Perlecan can also interact with growth factors or their binding proteins via its HS side chains. Some of these that are known to be expressed in ovaries that can bind HS include follistatin, a binding protein for activin, insulin-like growth factor binding protein 5, TGFß, and fibroblast growth factor-2 (FGF2) [52]. Thus, localization patterns showing FGF2 in the follicular basal lamina [53] is probably a reflection of the presence of HS in the basal lamina. These HS PGs are not merely passively acting to sequester growth factors or their binding proteins as perlecan has been shown to increase FGF2-mediated angiogenesis and mitogenesis in vitro [52].

Perlecan was also observed in the subendothelial basal lamina of the thecal vasculature but less in the basal laminas of arteriolar smooth muscle, as we observed previously for the ß1 chain of laminin [44]. It confirms the observations of Murdoch et al. [54] who localized perlecan within all vascular basal laminas in the major human tissues such as the kidney, liver, and heart. Perlecan was also localized to Call-Exner bodies that are aberrant basal lamina material located in the membrana granulosa and highly likely to be derived from granulosa cells [36]. Thus, it is likely that perlecan in these different basal laminas that are present in follicles represent different cellular sources of perlecan within the follicle.

Nidogen or entactin is not a PG but a sulfated glycoprotein that is an integral component of basal laminas. It has the capacity to associate with laminin and collagen type IV, and thus is thought to stabilize basal laminas. It was present in the follicular basal lamina in antral follicles, healthy, atretic, and regressed. However, little or undetectable staining was observed in primordial and small-antral follicles. Thus, nidogen is also developmentally regulated during follicular development. Its expression pattern parallels the increasing levels of laminin that occurs in follicles when they reach the antral stage [44]. Call-Exner bodies present in early and small-antral follicles also stained positively for nidogen, indicating that granulosa cells maybe the source of the nidogen in the follicular basal lamina. Nidogen was also immunolocalized to the subendothelial basal laminas of the ovarian vasculature (capillaries, venules, and arterioles) and to the basal lamina associated with arteriolar smooth muscle cells.

Versican probably has a multitude of functions because in different tissues it has a diverse range of expression patterns and it itself has different domains with different functions [55–57]. Even within tissues the expression pattern of versican is variable, sometimes in basal laminas, epithelia, or stroma, and often it is not uniformly expressed. Versican also has the ability to bind to a variety of extracellular matrix components, such as hyaluronin, fibulin 1, and L-selectin, and it is considered to be important in stabalizing extracellular matrix [58, 59]. Versican is also considered to be antiadhesive by the nature of it CS side chains, and it acts to stimulate cell migration and replication [60, 61]. Versican is developmentally regulated, and its expression is important for differentiation. In follicles versican was found to be developmentally expressed, not appearing until the antral stage when theca develops in bovine follicles. The thecal side of the follicular basal lamina was particularly enriched with versican, and the theca externa and membrana granulosa also contained versican. Whether the isoforms of versican in these areas are identical, and whether versican has many roles in follicular development remains to be determined.

Both versican and perlecan have been identified in human follicular fluid aspirated from follicles approaching ovulation following hCG injection [12]. Our studies show perlecan, which is a classical basal lamina component, to be localized to vascular basal laminas of the theca and to the follicular basal lamina. Thus, perlecan is probably not a true component of follicular fluid during follicular growth and development. It was possibly aspirated into the follicular fluid by the sampling procedure in the experiments where it was reported in follicular fluid [12], or alternatively, it only enters the follicular fluid on ovulation when the follicular basal lamina is degraded.

In summary, a number of PGs were identified and localized in the ovary. Their roles in follicle development have been implied from their known functions in other tissues, but these still need to be confirmed. The differential expression patterns of some of these suggest that they may have key roles in follicular development.

ACKNOWLEDGMENTS

We thank the following for their generous donation of antisera: Bruce Caterson, University of North Carolina at Chapel Hill; Larry Fisher, National Institute of Dental Research; Dieter Zimmermann and Maria Teresa Dours-Zimmermann, University of Zurich; Anne Underwood, CSIRO Molecular Science; and Marie Dziadek, University of Melbourne.

FOOTNOTES

First decision: 4 April 2000.

1 This work was supported by grants from the National Health and Medical Research Council of Australia, The Flinders Medical Centre Foundation, and The Flinders University of South Australia. Back

2 Correspondence. FAX: 61 8 8204 5450; ray.rodgers{at}flinders.edu.au Back

Accepted: May 2, 2000.

Received: February 29, 2000.

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K. Ndiaye, T. Fayad, D. W. Silversides, J. Sirois, and J. G. Lussier
Identification of Downregulated Messenger RNAs in Bovine Granulosa Cells of Dominant Follicles Following Stimulation with Human Chorionic Gonadotropin
Biol Reprod, August 1, 2005; 73(2): 324 - 333.
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J. S. Richards, I. Hernandez-Gonzalez, I. Gonzalez-Robayna, E. Teuling, Y. Lo, D. Boerboom, A. E. Falender, K. H. Doyle, R. G. LeBaron, V. Thompson, et al.
Regulated Expression of ADAMTS Family Members in Follicles and Cumulus Oocyte Complexes: Evidence for Specific and Redundant Patterns During Ovulation
Biol Reprod, May 1, 2005; 72(5): 1241 - 1255.
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J. Melrose, S. Smith, and J. Whitelock
Perlecan Immunolocalizes to Perichondrial Vessels and Canals in Human Fetal Cartilaginous Primordia in Early Vascular and Matrix Remodeling Events Associated with Diarthrodial Joint Development
J. Histochem. Cytochem., November 1, 2004; 52(11): 1405 - 1413.
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L. Mittaz, D.L. Russell, T. Wilson, M. Brasted, J. Tkalcevic, L.A. Salamonsen, P.J. Hertzog, and M.A. Pritchard<