Isolation and Characterization of Proteoglycans from Porcine Ovarian Follicular Fluid

Monomer proteoglycan was isolated from porcine ovarian follicular fluid by isopycnic CsCl centrifugation in the presence of 4 M guanidine HCl and protease inhibitors. The elution profile of the Dl preparation on Sepharose 2B was similar to that of monomer proteoglycan from bovine nasal cartilage, indicating a similar molecular size. Follicular fluid proteoglycans consist of about 20% protein, 50% dermatan sulfate, and 20% oligosaccharides rich in sialic acid, galactose, mannose, glucosamine, and galactosamine. The amino acid composition of this proteoglycan is significantly different from that of cartilage proteoglycans, with a higher proportion of aspartic acid, threonine, and lysine, and lower amounts of proline and glycine. Alkali-released dermatan sulfate chains are larger on Sepharose 6B (average M, = 56,000) than chondroitin sulfate chains from cartilage proteoglycans (average M, = 25,000), and iduronic acid accounts for 9% of total hexuronic acid. Disaccharide units released by chondroitinase ABC consists of 67% 4-sulfated, 22% 6-sulfated, 5% nonsulfated, and 5% disulfated disaccharides. After treatment with 0.05 M NaOH, 1 M NaBH4 at 45°C for 24 h, two major sialic acid-containing oligosaccharides were observed on Sephadex G-25, corresponding to pentaand hexasaccharides. The pentasaccharide contained sialic acid, galactose, glucosamine, and galactosamine in the proportions 1:2:1:1. The galactosamine is O-glycosidically linked to the protein core. This oligosaccharide accounts for approximately 77% of all the sialic acid in the follicular fluid proteoglycans. The hexasaccharide fraction contained sialic acid, galactose, mannose, and glucosamine in the proportions 1:2:1:2. It also contained a small amount of fucose and galactosamine. The linkage of these oligosaccharides to the protein core remains to be determined. The follicular fluid proteoglycans, unlike those from cartilage, do not interact with hyaluronic acid. Digestion with trypsin, chymotrypsin, or plasmin released dermatan sulfatepeptides nearly as small as those released by papain or alkali; in contrast, cartilage proteoglycans were resistant to plasmin and released peptides containing an average of more than four chondroitin sulfate chains after trypsin or chymotrypsin digestion.

After treatment with 0.05 M NaOH, 1 M NaBH4 at 45°C for 24 h, two major sialic acid-containing oligosaccharides were observed on Sephadex G-25, corresponding to pentaand hexasaccharides.
The pentasaccharide contained sialic acid, galactose, glucosamine, and galactosamine in the proportions 1:2:1:1. The galactosamine is O-glycosidically linked to the protein core. This oligosaccharide accounts for approximately 77% of all the sialic acid in the follicular fluid proteoglycans. The hexasaccharide fraction contained sialic acid, galactose, mannose, and glucosamine in the proportions 1:2:1:2. It also contained a small amount of fucose and galactosamine. The linkage of these oligosaccharides to the protein core remains to be determined. The follicular fluid proteoglycans, unlike those from cartilage, do not interact with hyaluronic acid. Digestion with trypsin, chymotrypsin, or plasmin released dermatan sulfatepeptides nearly as small as those released by papain or alkali; in contrast, cartilage proteoglycans were resistant to plasmin and released peptides containing an average of more than four chondroitin sulfate chains after trypsin or chymotrypsin digestion.
The presence of glycosaminoglycans in ovarian follicular * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
$ Visiting Fellow of National Institute of Child Health and Human Development. To whom requests for reprints should be addressed at the Department of Endocrinology, Toranomon Hospital, Akasaka Aoicho 2, Minatoku, Tokyo 107, Japan. fluid has been previously reported (l-3). These glycosaminoglycans have been partially characterized and their possible physiological roles, particularly with respect to ovulation, were discussed almost two decades ago (3). Jensen et al. (3) prepared glycosaminoglycans from a trypsin-digested follicular fluid by ethanol precipitation of trichloroacetic acid-soluble material and found that glycosaminoglycans constitute 0.2 to 0.3% (w/v) of the follicular fluid. They speculated that chondroitin sulfate and hyaluronic acid were the sources of the galactosamine and glucosamine. They also showed that the galactosamine/glucosamine ratio is smaller in the follicular fluid from small follicles, and that the average molecular weight of glycosaminoglycans from small follicles is higher, 98,000, than that from large follicles, 18,000, as estimated by osmometry. Gebauer et al. (4) recently investigated sulfated glycosaminoglycans in rat ovarian tissues and reported the presence of a heparin-like substance, chondroitin sulfate, and dermatan sulfate. Recent progress in the field of proteoglycan biochemistry has indicated that glycosaminoglycans are not present in tissues as free polysaccharide chains, but are covalently bound to core proteins as part of larger proteoglycan molecules which contain more than one polysaccharide chain. Therefore, the present study was undertaken to determine the characteristics of the native intact proteoglycan molecules to elucidate details about their structure and potential function in the ovarian follicles. The combined Dl-D2 fraction was further analyzed by a second CsCl equilibrium density gradient centrifugation, again under dissociative conditions but with a higher initial density (Fig. 1). The results of hexuronic acid, protein, and sialic acid measurements, amino acid analyses, and gel filtration on a Sepharose 2B column for each fraction are shown in Tables I  to III and Fig. 2. The hexuronic acid/protein ratio rapidly increases with the buoyant density of the material. The sialic acid/protein ratio also increases with the buoyant density of the material, but to a lesser extent than for hexuronic acid; and the sialic acid/protein ratio shows little change in D1,2-Dl through D1,2-D3 fractions. Gel filtration profiles show the presence of one major molecular species, containing hexuronic acid, protein, and sialic acid, that eluted as a broad peak, with K,, of 0.26 to 0.32. The K,, of these peaks decrease with increasing density, suggesting that higher density fractions contain molecules of larger hydrodynamic size. This material accounts for approximately 95% of the total hexuronic acid recovered in the Dl and D2 fraction of the first density gradient. Little or no difference was seen in the amino acid compositions of the bottom three fractions, D1,2-Dl through D1,2-D3, indicating that the protein in these fractions is similar, although the proportion of carbohydrate varies. This major molecular species markedly decreases in concentration in the upper two fractions, while another hexuronic acidcontaining component, which is smaller in molecular size, appears in the D1,2-D5 fraction. These findings suggest that the hexuronic acid-containing material recovered in the D3 and D4 fractions of the first density gradient (which accounts for up to 38% of the total hexuronic acid in the follicular fluid) differs from the major molecular species recovered from Fractions Dl and D2. The low buoyant density, smaller component was not studied further. Fig. 3 shows the elution profile of hexuronic acid from   Sepharose 2B chromatography for a Dl fraction isolated from medium-sized porcine ovarian follicles and that for a bovine nasal Dl preparation.
The Dl fractions from small and large follicles showed essentially the same profiles as did the Dl preparation from medium-sized follicles. Further, the Al preparation from medium-sized follicles showed essentially the same profile as did the Dl preparation; there was no evidence of aggregate formation as is observed for cartilage proteoglycans (23, 24). The elution profile suggests that the follicular fluid proteoglycans are polydisperse, with a KRv of 0.27, which is very close to that for cartilage proteoglycans (Fig. 3). Thus, the average molecular size of the follicular fluid proteoglycan is similar to that of hyaline cartilage proteoglycans.
An absorbance spectrum of the Dl preparation showed a higher extinction at 260 nm than at 280 nm, suggesting the presence of some nucleic acid. The Dl preparation was chromatographed on Sepharose 6B and UV absorption was measured for each fraction at 260 nm (Fig. 4). The void volume ( VO) peak contained hexuronic acid, protein, and sialic acid, while the peak at the column total volume (V,) did not. The chemical composition of the material at the peak in the void volume was essentially the same as that of the original Dl preparation, except for the absence of ribose in a neutral sugar analysis, further suggesting that the Dl sample contained some RNA (nucleic acid) contamination.
The amount was estimated as less than 5% of the dry weight of the Dl preparation from the absorbance spectrum and the known molar extinction coefficient of RNA (25). Small amounts of nucleic acid have been observed in Dl preparations isolated from aortic tissue by procedures similar to those used in this study (26,27).

Composition of Follicular Fluid Proteoglycan-The
chemical composition of the follicular fluid Dl fraction after Sepharose 6B chromatography to remove nucleic acid contamination is summarized in Table IV. The protein content is higher than that for proteoglycans from bovine nasal cartilage, and accounts for about 20% of the dry weight of the preparation. The amino acid composition also differs significantly from that reported for monomer proteoglycans isolated from cartilage (28): the follicular fluid proteoglycans contain more aspartic acid, threonine, and lysine, and less proline and glycine. Sepharose 6B chromatography of a Dl preparation after digestion with chondroitinase ABC indicated that essentially all the hexuronic acid was released from the core protein (Fig. 50). The presence of hexuronic acid and galactosamine in approximately equimolar concentrations also indicates the presence of chondroitin sulfate or dermatan sulfate.' A comparison of the carbazole/orcinol ratio (13-X), suggests that about 9% of the hexuronic acid is iduronic acid. After chondroitinase AC digestion under conditions which are known to degrade chondroitin sulfate almost completely (0.05 unit/mg of sample, 37"C, 1.5 h), followed by papain digestion to release the undigested dermatan sulfate chains from core protein (29), only 23% of the total hexuronic acid eluted at the column total volume ( V,) on a Sepharose 6B column (Fig. 5B). Even after digestion with excess chondroitinase AC (1 unit/mg of sample, 37"C, 3 h), followed by papain digestion, only 50% of the total hexuronic acid eluted at V, (Fig. 5C). The carbazole/orcinol ratio across the first peak ranges from 0.7 to 0.9, which "The term dermatan sulfate is used to indicate polysaccharide chains which contain both iduronic and glucuronic acid residues and not to indicate only those disaccharides in such chains which contain iduronic acid.
indicates that the iduronic acid/glucuronic acid ratio is higher than in the intact chains, but these partially digested fragments still contain a high proportion of glucuronic acid, consistent with the fact that only about 9% of the total is iduronic acid. This is probably due to the fact that chondroitinase AC The arrow near V, shows the elution position for protein were analyzed for hexuronic acid by both the carbazole and orcinol procedures. The ratio was constant across the peak (average 0.89) within experimental error, indicating that the proportion of iduronic acid to glucuronic acid in the chains was independent of chain size (Fig. 5A). The chondroitinase AC-resistant fragments, indicative of the presence of iduronic acid, are smaller than undigested chains which can be released by papain or alkali treatment (29). This suggests that the dermatan sulfate is a co-polymer with iduronic acid and glucuronic acid residues in the same chain, as in the case of aortic proteoglycans, knee joint cartilage, umbilical cord, and skin dermatan sulfate (31-36). The average molecular size of the dermatan sulfate chains in the Dl preparation was determined by chromatography of papain-or alkali-treated Dl samples on Sepharose 6B (Fig. 5A). The hexuronic acid eluted from the column as a rather broad peak, with an average K,, of 0.30. This K,, would correspond to an apparent molecular weight of 56,000, provided the Sepharose 6B used in these  Wasteson (37). This estimate agrees well with the ratio of xylose to hexuronic acid in the Dl samples, approximately 1: 110 (Table IV), which indicates that the average number of the repeating disaccharide units per chain would be about 110, corresponding to a molecular weight of about 51,000. Based on the hexuronic acid content of the Dl preparation, the sodium salt of the dermatan sulfate should be approximately 50% of the dry weight of the sample.

chondroitinase
ABC by Sepharose 6B chromatography (Fig.  7B). The peak material was isolated and its composition determined. The ratios of protein to sialic acid, glucosamine, galactose, and xylose did not change significantly from the original Dl preparation (Table IV). Alkaline borohydride treatment of this protein core preparation (9) converted ap-Paper chromatography was used to separate the unsaturated disaccharides generated by digestion of the Dl preparation with chondroitinase ABC. The results (Fig. 6) indicate that 67% of the disaccharides were 4-sulfated, 22% 6-sulfated, and 5% nonsulfated. A small peak between the origin and the 6-sulfated disaccharide suggests the presence of disulfated disaccharides (19), which account for about 5% of the total, and probably contain iduronic acid (38). However, which fractions contain iduronic acid remains to be determined. The glucose present in the Dl preparation might have been derived from small amounts of glycogen, which have been shown to be present in human ovarian follicles by histochemical techniques (39).

Characterization of Protein Core Preparation and Sialic Acid-containing
Oligosaccharides-The follicular fluid Dl preparation contained approximately 6% sialic acid by weight. On Sepharose 2B chromatography of the Dl preparation, the elution profile of sialic acid was essentially identical with those of hexuronic acid and protein (Fig. 3). Further, the chromatogram for the Dl preparation after chondroitinase ABC digestion shows that the sialic acid co-elutes on Sepharose 2B with protein (K," = 0.52), except for the small protein peak which comes from the chondroitinase ABC preparation (Fig. 7A). These data indicate that the sialic acid-containing material is associated with the proteoglycan molecules and is resistant to chondroitinase ABC digestion. A protein core preparation was isolated after removing the dermatan sulfate chains with Gel P-10 of alkaline borohydride-treated follicular fluid proteoglycan (Dl). The eluent was analyzed for sialic acid (---) and hexose (---). Lettered areas designate fractions which were pooled for further study. Anthrone peak at VO contains dermatan sulfate, and the V, peak is due to nonspecific color reaction by salt.
proximately 85% of the galactosamine residues into galactosaminitol residues; the galactosamine/glucosamine ratio changed from 0.89 to 0.13 by alkaline borohydride treatment, suggesting that these galactosamine residues were attached to the protein core of the proteoglycan by 0-glycosidic bonds. The remaining nonreduced galactosamine residues probably are derived from residual repeat disaccharides near the linkage regions of the dermatan sulfate chains, which resist chondroitinase ABC digestion (40).
Dl samples were treated with alkaline borohydride, chromatographed on Sephadex G-75, and the elution profiles of hexuronic acid and sialic acid determined (Fig. 8A). Essentially all the hexuronic acid eluted in the void volume, and all the sialic acid-containing materials were released by this treatment (K,, = 0.92). Dl samples were also treated with papain, chromatographed on Sephadex G-75, and the elution profiles for hexuronic acid and sialic acid were determined (Fig. 8B). Again, the uranic acid-containing peak was excluded by this column, while the sialic acid eluted as a very broad included peak (K,, = 0.48). Since the sialic acid peak elutes earlier after papain than after alkaline borohydride treatment, this indicates that papain digestion of the Dl preparation releases peptides which contain a number of sialic acid-containing oligosaccharides.
The molecular sizes of the sialic acid-containing oligosaccharides were determined by Sephadex G-25 chromatography after alkaline borohydride treatment of the Dl preparation.
Two major peaks were observed (Fig. 9A), and the molecular sizes of these oligosaccharides were estimated by use of co-chromatography with "H-labeled oligosaccharides of hyaluronic acid. A major sialic acid-containing peak eluted between tetra (HA,)-and hexa (HAe)-saccharides, and a minor sialic acid-containing peak eluted between HAs and HAa. Sufficient amounts of these oligosaccharides for the chemical analyses were isolated from an alkaline borohydridetreated Dl preparation by chromatography on a Bio-Gel P-10 column (0.7 X 110 cm), eluted with 0.1 M pyridinium acetate buffer, pH 5.0. Peak fractions were collected and lyophilized directly (Fig. 9B). The results of sialic acid, hexosamine, hexosaminitol, and neutral sugar analyses are shown in Table  V. The major sialic acid-containing oligosaccharide, eluting as a pentasaccharide, accounted for 77% of all the sialic acid. It contained sialic acid, galactose, galactosaminitol, and glucosamine in the proportions 1:2:1:1. The data suggest that this oligosaccharide is a pentasaccharide with galactosamine as the linkage sugar to the protein core by an 0-glycosidic bond. The minor sialic acid-containing oligosaccharide, eluting between hexa-and octasaccharide, accounted for 23% of the total sialic acid. It contained sialic acid, mannose, galactose, and glucosamine in the proportions 1:1:2:2, suggesting a hexasaccharide. The discrepancy of the elution position might be due to the molecular size of sialic acid, or the hydrodynamic conformation of the oligosaccharide slightly differs from that Essentially the same hexuranic acid elution profile was obtained after papain digestion and treatment with alkali, conditions which are known to release single chondroitin sulfate chains from cartilage proteoglycan (29). The relatively broad peak centered at KIly = 0.71 indicates the polydispersity of dermatan sulfate chains (Fig.  1OA). The hexuronic acid elution profiles observed after either trypsin, chymotrypsin, or plasmin digestion were essentially identical, with a K,, of about 0.67 (Fig. 10, B to D). The dermatan sulfate-peptide fragments released by these enzymes are therefore only slightly larger than those released by papain. These results suggest that the individual dermatan sulfate chains of follicular fluid proteoglycan are separated from each other by peptide regions which are susceptible to these various specific proteases. chains attached, while trypsin alone yields peptides with even more chondroitin sulfate chains attached (41). Further, bovine nasal Al or Dl proteoglycan samples were not significantly affected by plasmin digestion under conditions identical with those used for the follicular fluid Dl (Fig. 1OD).

Interaction of Follicular
Fluid Proteoglycans with Hyaluronic Acid-The follicular fluid Dl preparation was incubated with hyaluronic acid and then chromatographed on Sepharose 2B. The hexuronic acid elution profile was essentially identical with that for the Dl preparation without hyaluronic acid, indicating that these proteoglycans, in contrast to cartilage proteoglycans, did not interact with hyaluronic acid (Fig. 11). No hyaluronic acid was found in any of the follicular fluid fractions, Dl through D4, using the hyaluronic acid assay described under "Experimental Procedures." The sensitivity of the assay as determined by use of hyaluronic acid standards was such that the presence of 0.12 I-18 of hyaluronic acid/100 pg of Dl (or D2, D3, D4) samples would have been detected. DISCUSSION The porcine ovarian follicular fluid proteoglycans showed a disperse buoyant density distribution in the dissociative CsCl density gradient, which is mainly due to the heterogeneity in the amounts of dermatan sulfate chains per molecule. As in the case of cartilage proteoglycans, the ratio of glycosaminoglycan to protein largely determined the buoyant density and the molecular size of follicular fluid proteoglycans (42). On the other hand, the amounts of the oligosaccharides which contained sialic acid are relatively constant with the buoyant density of the molecule. Accordingly, the galactosamine/glucosamine ratio, which reflects the ratio of the dermatan sulfate to the oligosaccharides, decreased in the lower buoyant density fractions. This is also analogous to cartilage proteoglycans, where the chondroitin sulfate/keratan sulfate ratio in monomers decreases in the lower buoyant fractions of CsCl density gradients (42).
The data presented here suggest that the proteoglycans isolated from porcine ovarian follicles differ in many respects from the well characterized proteoglycans from cartilage, although both are of similar average hydrodynamic size. The follicular fluid proteoglycans contain dermatan sulfate chains, which have an average molecular weight of 56,000, which is more than twice as large as that of chondroitin sulfate chains from cartilage proteoglycans (Mr z 25,000) (42), and are larger than most chondroitin or dermatan sulfate chains reported to date (43). The protein core of the follicular fluid proteoglycans after chondroitinase ABC digestion appears to be larger (44, 45), has a different amino acid composition, and most interestingly, exhibits a different susceptibility to enzymatic digestion than the corresponding core protein derived from cartilage proteoglycan.
The follicular fluid proteoglycans do not interact specifically with hyaluronic acid to form the high molecular weight complexes which are responsible for aggregate formation by cartilage proteoglycans. Approximately 20% of the dry weight of the follicular fluid proteoglycan consists of two major oligosaccharides which contain sialic acid. There have been no previous reports that either of these oligosaccharides are present in cartilage or other proteoglycans. Skeletal keratan sulfate, which is frequently found in cartilage and aortic proteoglycan (31, 46), was not found in the follicular fluid proteoglycans, although the pentasaccharide could have a similar structure to the linkage region between keratan sulfate and the core protein of proteoglycans.
Since the follicular fluid proteoglycans have a similar hydrodynamic size as cartilage proteoglycans, they are likely to have similar molecular weights as well, around 2 to 2.5 x 10" (28). This suggests that the follicular fluid proteoglycan has a core protein of molecular weight about 400,000 with an average of about 20 dermatan sulfate chains and 350 sialic acid-containing oligosaccharides attached.
The composition of the major oligosaccharide component (pentasaccharide) of the follicular fluid proteoglycan resembles that of some of the oligosaccharides found in various glycoproteins, such as the blood group-specific glycoprotein isolated from human or porcine ovarian cyst fluid, gastric, and submaxillary mucins (47). The oligosaccharides found in these glycoproteins are very heterogeneous, consist mainly of variable amounts of IV-acetylgalactosamine, N-acetylglucosamine, galactose, and fucose, have IV-acetylgalactosamine as a linkage sugar to hydroxyl groups of serine or threonine and show blood group specificities by the substitution of "precursor" or core oligosaccharides with the specific sugar residues (48). One of the precursor tetrasaccharides found in a blood groupspecific glycoprotein from human ovarian cyst fluid, namely D-GalPl ---f 3 [D-Gal/31 + 4 D-GlcNAcPl --+ 61 D-GalNAc (49), has the same composition as the pentasaccharide of follicular fluid proteoglycan except for the absence of a sialic acid residue. Both the blood group-specific glycoprotein and the proteoglycan are derived from ovarian tissue. The glycoprotein has been found in pathological ovarian cyst fluid, but not in normal follicles. These two materials have similar oligosaccharide composition, but differ in many respects (50). The blood group-specific glycoproteins have more highly heterogeneous oligosaccharides as their main component, do not contain glycosaminoglycans, and have a different amino acid composition.
A recent report on the characterization of the blood group-specific glycoprotein from human ovarian cyst fluid by Bhaskar et al. (50) suggests that cyst fluid contains little or no proteoglycan of the type described in this paper.