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Originally published In Press as doi:10.1074/jbc.M606029200 on January 3, 2007

J. Biol. Chem., Vol. 282, Issue 11, 8011-8018, March 16, 2007
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Identification of a Novel Chondroitin-sulfated Collagen in the Membrane Separating Theca and Granulosa Cells in Chicken Ovarian Follicles

THE GRANULOSA-THECA CELL INTERFACE IS NOT A BONA FIDE BASEMENT MEMBRANE*

Susanna Hummel{ddagger}1, Sabine Christian{ddagger}1, Andreas Osanger{ddagger}, Hans Heid§, Johannes Nimpf{ddagger}, and Wolfgang J. Schneider{ddagger}2

From the {ddagger}Department of Medical Biochemistry, Max F. Perutz Laboratories, Medical University of Vienna, Dr. Bohr Gasse 9/2, A-1030 Vienna, Austria and the §Deutsches Krebsforschungszentrum Heidelberg, D-69120 Heidelberg, Germany

Received for publication, June 23, 2006 , and in revised form, November 13, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The membranous structure separating the granulosa from theca cells in the developing ovarian follicles of birds is generally perceived as a genuine basement membrane (BM). Previously, we suggested that this membrane is unusual in that it lacks several typical BM components, e.g. collagen IV, laminin B, perlecan, and fibronectin (Hummel, S., Osanger, A., Bajari, T. M., Balasubramani, M., Halfter, W., Nimpf, J., and Schneider, W. J. (2004) J. Biol. Chem. 279, 23486–23494). We have now identified a novel chondroitin sulfate-modified collagen, tentatively termed ggBM1 (Gallus gallus basement membrane protein1) as a major component of the border between the vascularized theca and the epitheloid granulosa cells. In biosynthetic experiments using [3H]proline and [35S]sulfate, ggBM1 was shown to be synthesized by and secreted from the granulosa cells that support the developing oocyte. The acidic heterogeneous 135-kDa proteoglycan was converted to a protein with an apparent Mr of 95,000 by treatment with chondroitinase ABC and was completely degraded by collagenase. Sequencing of tryptic fragments revealed peptides typical of collagens. The follicular BM accumulated apolipoprotein B and apo-VLDLII, the major resident proteins of the yolk precursor very low density lipoprotein. Interestingly, and likely indicating an analogous situation to the follicle, ggBM1 is also a component of Bruch's membrane of the eye, which separates the vascularized choroid from retinal pigmented epithelial cells. Based on our data we propose that in addition to thecal perlecan, ggBM1 is involved in the transfer of yolk precursors from the thecal capillary bed to oocyte surface lipoprotein receptors mediating their uptake into oocytes.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
During the development of ovarian follicles in oviparous species, the formation and maintenance of extracellular matrices and laminar structures for mechanical support are important processes. The coordinated regulation of the synthesis of their components and assembly is one of the prerequisites for the normal growth of the follicles' central giant germ cell, the oocyte. In the final 7-day period of rapid growth of the chicken oocyte, it is surrounded by a concentric arrangement, from the periphery inwards, of (i) vascularized connective tissue representing an extension of the follicle stalk, (ii) the theca proper, which consists of the theca externa, a broad layer of stratified cells with embedded capillaries, and the narrower theca interna, (iii) an acellular layer commonly designated as basal lamina (1, 2) or basement membrane (3, 4) that functions in follicle compartmentalization, (iv) an epitheloid layer of granulosa cells, and (v) the zona pellucida. Previous studies (5, 6) have demonstrated that oocyte growth and development are achieved through the unprecedentedly efficient uptake of hepatically synthesized yolk precursor proteins by multifunctional oocytic plasma membrane receptors belonging to the low density lipoprotein receptor (LR)3 gene family. We have shown that at least two such receptors, termed LR8 (7–9) and LRP380 (10), mediate yolk formation via uptake from the serum compartment of macromolecules including very low density lipoprotein (VLDL), vitellogenin, {alpha}2-macroglobulin, complement component C3, and riboflavin-binding protein (11–13). The size of serum-borne VLDL particles, in the order of 40-nm diameter (14, 15), poses the question as to how these large complex molecules gain access to the oocytic plasma membrane in the core of the follicle. Detailed ultrastructural studies in the late 1970s (16) established that the endothelium of capillaries within the theca layer shows extensive fenestrae with widths of up to 50 nm, many of which appear to provide direct access from the lumen to the pericapillary space. Thus, when macromolecules are released from the capillaries, they first encounter extracellular matrices, from where they diffuse across the basement membrane, through gaps between granulosa cells and past the zona pellucida, to home in on the above described high affinity endocytic receptors in the oocyte plasma membrane. The membranous structure separating granulosa cells from theca interna has been shown to be permeable to serum-derived VLDL particles (2, 4). These and other studies suggested that the high concentration of the particles residing within the membrane, which we term "basement membrane" (BM) in this report, is related to its capacity to become saturated with VLDL, thus facilitating penetration of the particles in the fluid phase to the oocyte surface (4).

While it is widely assumed that the BM is composed of the typical BM components, our factual knowledge about the composition of this membrane is very limited (2, 15, 17). The above considerations raise the possibility that negatively charged macromolecular structures (e.g. acidic proteoglycans) interact with the positively charged apolipoprotein B-100, the major protein component of VLDL. Thus, based on the generally accepted fact that the heparan sulfate proteoglycan, perlecan, is an important functional component of typical basement membranes, we have recently molecularly characterized chicken perlecan (18). One of the domains of chicken perlecan, which contains a cluster of four lipoprotein binding repeats typical of the LR gene family, indeed interacts with serum VLDL (18). Surprisingly, while perlecan has been well conserved in a wide range of species, and is synthesized by theca cells, it clearly is absent from the follicular BM (18). Agrin, laminin B, fibronectin, and syndecan-1/syndecan-3 are also not found in this membrane. Perlecan likely constitutes a transient storage site for VLDL in the thecal matrix, where its multistep pathway for ultimate uptake into the oocytic yolk compartment is initiated (18).

Thus, studies to date have not led to the unambiguous identification of any of the molecules constituting the follicular BM. In the current study, we have isolated this unique matrix and addressed its biochemical characterization. We show that the main component is a chondroitin sulfate-modified collagen, possibly related to type IX collagen, which is synthesized by and secreted from granulosa cells. The origin and properties of this molecule provide an explanation for the mechanical strength and plasticity of the BM during oocyte growth as well as for its ability to interact with VLDL.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Experimental Animals and Antibody Preparation—Mature Derco-brown laying hens (Heindl Co., Vienna, Austria) and quail hens (Coturnix coturnix) were maintained on layer's mash with free access to feed and water under a daily light period of 16 h. Animals were sacrificed by decapitation. Antibodies were produced in adult female New Zealand White rabbits (18). All procedures were performed according to the protocols approved by the Animal Care Committee of the Medical University of Vienna. Rabbit antisera used have been previously described in the indicated references: anti-ggBM1, directed against the SDS-PAGE-isolated major follicle BM component, and anti-chicken perlecan, directed against perlecan LA repeats 2–4 (18), anti-ZP1 (19), anti-chicken apo-VLDLII (20), and anti-chicken apoB-100 (14). Antiserum directed against a 50-kDa apoB-fragment (yB5) present in VLDL isolated from yolk (14) was raised against the gel-eluted protein.

Isolation of Granulosa Cell (GC) Sheets, Theca, and Basement Membrane—Preovulatory follicles (F1 to F3) were dissected from adult laying hens or quails immediately after decapitation. Follicles were punctured with sterile forceps, yolk was squeezed out, and the GC sheets were extruded. The sheets were washed several times in 10 ml of PBS (80 mM Na2HPO4, 20 mM NaHPO4, 100 mM NaCl) until the residual yolk was removed and subsequently incubated in buffer containing 0.5 M Tris, pH 6.8, and 5 mM ETDA for 30 min at room temperature. The BM and the zona pellucida (ZP) were separated mechanically under a light-microscope using sharp forceps. The ZP was sedimented by centrifugation at 5,000 x g for 5 min. The two membranes were dissolved in SDS-PAGE sample buffer containing 50 mM dithiothreitol and were stored at –20 °C. The residual follicular thecal portion was washed in PBS, transferred to 5 ml of 8 M urea, and homogenized for 1 min using an Ultra Turrax T25 homogenizer. The homogenate was incubated at room temperature overnight and was then centrifuged (13,000 x g, 10 min) to remove debris and non-dissolved material.

Enzymatic Treatment of ggBM1 Protein—The isolated BM or ZP (as indicated) were incubated with heparinase I (from Flavobacterium heparinum; Sigma catalog number H2519), heparinase III (from F. heparinum; Sigma catalog number H8891), chondroitinase ABC (from Proteus vulgaris, Sigma catalog number C2905), hyaluronidase (from bovine testes or from Streptomyces hyalurolyticus; Calbiochem catalog numbers 385931 and 389561), collagenase IV (from Clostridium histolyticum; Sigma catalog number C5138) or in buffer alone according to the manufacturer's recommendations. Following incubations, the samples were dissolved in SDS-PAGE sample buffer containing 50 mM dithiothreitol, heated to 95 °C for 5 min, and subjected to SDS-PAGE.

Metabolic Labeling of GC Sheets with Na235SO4 or with L-[2,3,4,5-3H]Proline and Immunoprecipitation—For metabolic labeling, freshly isolated GC sheets were incubated in Dulbecco's modified Eagle's medium (Invitrogen) containing 120 µCi/ml Na235SO4 (PerkinElmer Life Sciences) or 250 µCi/ml L-[2,3,4,5-3H]proline (Amersham Biosciences) for 24 h at 37 °C, 5% CO2. The BM was separated from the ZP as described above. For immunoprecipitation, 750 µl of medium were incubated with 10 µl anti-ggBM1 antiserum or preimmuneserum for 4 h at 4 °C. 25 µl of a 50% (w/v) suspension of protein-A-Sepharose (Amersham Biosciences) in PBS were added, and incubation was continued overnight. The beads were washed four times with 500 µl of PBS + 1% Triton X-100, resuspended in 40 µl of SDS-PAGE loading buffer, and cleared by centrifugation, and the supernatant was used for electrophoresis.

SDS-PAGE and Western Blot Analysis—Proteins were subjected to one-dimensional SDS-PAGE under reducing conditions. Molecular weights were estimated by use of a molecular weight marker (SDS molecular weight markers Broad Range or Precision Plus Protein Standards, Bio-Rad). The gels were either stained with Coomassie Blue (Gelcode-blue; Pierce) or with Alcian blue (21). For Western blot analysis, the proteins were transferred to a nitrocellulose membrane (Hybond-C; Amersham Biosciences) by semidry blotting. After incubation with 5% nonfat milk powder in PBS plus 0.1% Tween 20 (blocking solution), membranes were incubated with primary antisera against ggBM1 (1:500), perlecan 2–4 (1:500), or apolipoprotein B-100 (1:500) in blocking solution. Blots were then incubated with horseradish peroxidase-conjugated goat anti-rabbit secondary antibodies (1:50,000 dilution, Sigma), and signals detected using an enhanced chemiluminescence system (ECL, Pierce) according to the manufacturer's instructions.

Immunohistochemistry—For cryosections, small yellow follicles and eyes were embedded in freezing reagent (Microm Austria) and immediately frozen. Cryostat sections of 20 µm were prepared and fixed on SuperFrostR Plus slides (Menzel-Gläser, Braunschweig, Germany) with acetone/methanol (1:1) for 10 min. For paraffin sections, tissues were fixed in 4% formaldehyde and embedded in paraffin. Sections of 8 µm were prepared and deparaffinized in XEM-200 (Vogel Co.) by gentle shaking for 20 min. The sections were rehydrated using increasing dilutions of ethanol. In both cases, the slides were blocked with 1% goat serum, 3% BSA in PBS for 1 h at room temperature, and were then incubated with antisera against ggBM1 (1:250), ZP1 (1:5.000), perlecan 2–4 (1:250), apolipoprotein B-100 (1:250), apo-yB5 (1:250), or apoVLDLII (1:250) as indicated, for 1 h or overnight in a humid chamber. For immunofluorescence microscopy, sections were rinsed three times in PBS and incubated with fluorescence-labeled goat anti-rabbit secondary antibodies (Alexa Fluor-488 at 1:500 dilution, Molecular Probes) for 2 h. Counterstaining of cell nuclei was performed with DAPI. Specimens were mounted in fluorescent mounting media (DAKO) and analyzed by confocal microscopy (Zeiss, LSM 510Meta).

Determination of Partial Protein Sequence of ggBM1—For protein sequencing, chondroitinase ABC-digested BM (100 µg of protein) was subjected to 8% SDS-PAGE under reducing conditions. To obtain specific peptide fragments of ggBM1, the protein was visualized with Coomassie Blue, transferred to Immobilon polyvinylidene difluoride membrane (Millipore, Schwalbach, Germany), and digested with sequencing grade modified trypsin (Promega, Mannheim, Germany) directly on the membrane according to (22). The peptides obtained were separated by high pressure liquid chromatography (HPLC) using a Hypersil C18 BDS 3-m LC-Packings column (150 x 1.0 mm) and a 130 A HPLC separation system (Applied Biosystems, Darmstadt, Germany) with mobile phases consisting of solvent A (water, 0.1% trifluoric acid) and solvent B (80% acetonitrile, 0.085% trifluoric acid). HPLC-separated fragments were sequenced on polybrene-treated filters with a Procise 494 A protein sequencer from Applied Biosystems.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Analysis of the Extracellular Membranes of the Chicken Ovarian Follicle—Both the ZP and the BM were obtained from fragments of sheets of GC isolated from follicles in the size range of 1 to 2.5 cm diameter (the F5, F4, F3, and F2 follicles) according to Ref. 23. Following incubation in 5 mM EDTA for 30 min at 23 °C, the translucent basement membrane was retrieved with sharp forceps, and the zona pellucida together with GC debris were sedimented by centrifugation. The protein contents of the two membranes were analyzed by SDS-PAGE (Fig. 1). Staining with Coomassie Blue (lanes 1 and 2) revealed that there were very few, if any, comigrating bands in the two preparations, indicating their complete separation. The ZP (lane 1) showed the typical protein components characterized previously (24). In the BM (lane 2), a diffuse band at 135 kDa was the predominant component. Bands in the range of 200 and 165 kDa, and of approximately 68 and 84 kDa, respectively, which are further described below, varied in intensity in different BM preparations. Staining of the gel with Alcian blue (lanes 3 and 4), which binds to highly negatively charged macromolecules (21), revealed that the only visualized component in the BM (lane 4) was the component of approximately 135 kDa, designated ggBM1 (Gallus gallus BM protein 1). In the ZP preparation, a diffuse band of ~210 kDa, not seen with Coomassie Blue, was stained. The diffuse migration and binding of Alcian Blue of these components indicated that they were negatively charged proteoglycans. Indeed, the 210-kDa protein in the ZP preparation has been identified as an ovomucin (25).4


Figure 1
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  		FIGURE 1.
Analysis of the follicular extracellular membranes. Zona pellucida (lanes 1 and 3) and basement membrane (lanes 2 and 4) were isolated and dissolved as described under "Experimental Procedures." Aliquots (10 µg of protein/lane) were subjected to 8% SDS-PAGE under reducing conditions. The gels were stained with Coomassie Blue (lanes 1 and 2) or Alcian blue (lanes 3 and 4). Molecular weight markers are indicated.

 
To test for the presence of covalently attached carbohydrates in the 135-kDa ggBM1, we treated aliquots of isolated BM with a set of glycosidases specific for polyanionic polysaccharides. As shown in Fig. 2A, treatment with chondroitinase ABC from Proteus vulgaris, which liberates side chains from chondroitin sulfates A and C, and digests hyaluronan side chains, led to disappearance of the diffuse 135-kDa band, and generated a narrow band with an apparent size of approximately 95 kDa (compare lanes 3 and 4). The same result was obtained by treatment with hyaluronidase from bovine testis, which cleaves beta-N-acetylhexosaminine glycosidic bonds in hyaluronic acid, chondroitin, and chondroitin sulfates (lane 5). Heparinase I (lane 1) and III (lane 2) had no discernible effect on ggBM1. Thus, ggBM could carry either chondroitin sulfate or hyaluronic acid. To distinguish between these possibilities, BM was treated with hyaluronidase from S. hyalurolyticus, which recognizes and degrades only hyaluronic acids (lane 6). As this enzyme had no effect on ggBM1, at least under our conditions, the data strongly suggest that ggBM1 is a chondroitin sulfate-containing proteoglycan. The proteins at 200, 165, 84 (prominent in the samples in lanes 3–6), and 68 kDa (cf. Fig. 1) remained unaffected by the various enzyme treatments. Due to the close relationship between quail (C. coturnix) and chicken, both species are members of the phasianidae family, we were interested in a comparison of their BM components. Fig. 2B shows the similarity between the major ZP protein ZP1 (Ref. 19; lanes 1 and 2) and the BM proteins (lanes 3 and 4) from the two avian species. Despite considerable technical difficulty in isolating the BM from the smaller quail follicles, it can be seen that the diffuse band is only slightly larger than ggBM1, and the abovementioned bands at 200 and 165 kDa (lane 3) are present. Western blotting of BMs from chicken and quail follicles with anti-ggBM1 antibody revealed crossreactivity with the quail protein (Fig. 2C). Thus, the composition of the follicular BM of the quail, and particularly the component corresponding to ggBM1, is likely equivalent to that of the chicken.


Figure 2
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  		FIGURE 2.
Enzymatic treatment of the follicular basement membrane and comparison of follicular membranes of chicken and quail. A, follicular BM was isolated as described under "Experimental Procedures". Aliquots were treated with heparinase I (lane 1), heparinase III (lane 2), chondroitinase ABC (lane 4), hyaluronidase from bovine testis (lane 5), and hyaluronidase from S. hyalurolyticus (lane 6); lane 3 contained untreated extract. The samples (10 µg/lane) were subjected to 8% SDS-PAGE under reducing conditions, and the gel was stained with Coomassie Blue. Molecular weight markers are indicated. B, ZP (lanes 1 and 2) and BM (lanes 3 and 4) of chicken (lanes 1 and 3) and quail (lanes 2 and 4) follicles were isolated and dissolved as described under "Experimental Procedures." Aliquots (10 µg of protein/lane) were subjected to 8% SDS-PAGE under reducing conditions. The gels were stained with Coomassie Blue. Molecular weight markers are indicated. C, aliquots (10 µg of protein/lane) of BM from chicken (lane 1) and quail (lane 2) follicles were subjected to 10% SDS-PAGE under reducing conditions and Western blotting with anti-ggBM1 antiserum as described under "Experimental Procedures." Molecular weight markers are indicated.

 


Figure 3
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  		FIGURE 3.
Analysis of enzymatic treatment of follicular membranes. A, ZP (lane 1) and BM (lanes 2–4) were isolated, and aliquots of BM were digested with chondroitinase ABC (lane 3), hyaluronidase from bovine testis (lane 4), or incubated in buffer alone (lane 2) as described under "Experimental Procedures." The samples (10 µg of protein/lane) were subjected to 8% SDS-PAGE under reducing conditions and subsequent Western blot analysis using anti-ggBM1 antiserum (1:500 dilution). Horseradish peroxidase-conjugated goat anti-rabbit secondary antibodies were used at 1:50,000 dilution. Molecular weight markers are indicated. B, aliquots of the BM were incubated in buffer alone (lane 1) or in buffer containing collagenase IV (lane 2). Samples (10 µg of protein/lane) were subjected to 8% SDS-PAGE under reducing conditions. The gel was stained with Coomassie Blue. Molecular weight markers are indicated.

 
Characterization of ggBM1—Next, to investigate the molecular nature of ggBM1, we obtained protein sequence data. To this end, we digested BM with chondroitinase ABC and analyzed the 95-kDa ggBM1 protein after transfer to Immobilon polyvinylidene difluoride membrane as described under "Experimental Procedures." Since the amino terminus was blocked, tryptic fragments were subjected to conventional sequencing, resulting in four clearly collagen-derived peptides containing the characteristic Gly-Pro-Pro (GPP) repeats, K/RGPPhG/APPhGPPhGPPhG, K/RGPPhGPPhGPPhGPPhGEE, K/RGPPhGIPhGHPhGPPhGAPhGLL, and K/RGPPhGIPhGAPhGPPhGAPhGLL, where K/R are the hypothetical aminoterminal residues, and Ph denotes chemically identified hydroxyproline residues. We did not find non-collagenous sequences, even using digestions of ggBM1 with different proteases. Thus, based on the enormous number of highly homologous sequences in the large population of collagen genes, the design of specific oligonucleotides toward molecular characterization of the cDNA specifying ggBM1 was judged impossible. We also were unsuccessful to obtain clearly identifiable clones by screening a follicle expression library with affinity-purified anti-ggBM1 IgG. However, extensive multispecies BLAST searches as well as analysis of the chicken genome data base (version 38.1n) indicated that the ggBM1 band contains polypeptide(s) similar or identical to chicken type IX (preferentially its {alpha}2 chain) and/or type II collagens.

To gain further insight into the nature and localization of ggBM1, we raised a rabbit antiserum against untreated ggBM1 as antigen. Fig. 3 shows that anti-ggBM1 indeed recognizes ggBM1 in BM extracts with high specificity, but not in ZP (A, lanes 1 and 2), and also reacts with ggBM1 following enzymatic removal of the chondroitin sulfate chains (lanes 3 and 4 and cf. Fig. 2). In the enzyme-treated samples, not only the 95-kDa band, but also bands at approximately 84 (lanes 3 and 4) and 68 kDa (lane 4) showed immunoreactivity. Previously, purified [14C]glycine-labeled type IX collagen from chicken embryo sternal cartilage has been reported to show a diffuse chondroitinase ABC-sensitive larger band, as well as 84 and 68 kDa apparently non-glycated polypeptides under reducing conditions (26). Fig. 3B demonstrates that ggBM1 is completely degraded by incubation of BM extracts with collagenase, which also obliterates the 200- and 165-kDa bands, strongly supporting the notion of ggBM1 being a chondroitin-sulfated collagen.


Figure 4
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  		FIGURE 4.
Localization of ggBM1 in the chicken follicle and eye. Paraffin sections of chicken follicles, and cryosection of chicken embryo eyes were prepared and treated as described under "Experimental Procedures." A, Alcian blue stain of a chicken follicle. B and C, immunohistochemistry of a chicken follicle (B) and a chicken eye (C) using anti-ggBM1 antiserum (1:500 dilution). Cell nuclei were counterstained with DAPI (B). R, retina; b.l., basal lamina.

 
Next, immunohistochemistry performed with the anti-ggBM1 antibody (Fig. 4) demonstrated the exclusive localization of this molecule to the BM. The antibody stained the membrane forming the boundary between the GC and the vascularized theca interna (B), congruent with the staining of a follicle section observed with Alcian Blue (A). In analogy, immunostaining of a section through the eye of a chicken embryo (C) revealed the presence of ggBM1 mainly in Bruch's membrane separating the vascularized choroid from the pigmented epithelium of the retina (27). The inner limiting membrane or basal lamina (28) did not contain significant amounts of ggBM1. Furthermore, ggBM1 appears to be quite specific to the follicles and the eye, since other organs (kidney, testis, liver) showed no detectable immunohistochemical staining (data not shown).

Biosynthesis of ggBM1—Since ggBM1 in the follicle was located on the basal aspect of the GC, which in maturing follicles form a monolayer of epitheloid cells separated from the oocyte by the ZP, GC were the likely source of ggBM1. Thus, to directly analyze the capacity of GC to synthesize ggBM1, we performed the biosynthetic labeling experiments described in Fig. 5. As the results demonstrate, the biosynthetically active GC sheets (29) incorporated [35S]sulfate as well as [3H]proline into synthetic products. Following continuous labeling with either of the radioactive precursors for 24 h, the medium was removed, and the GC sheet manipulated to separate and isolate the ZP and BM, respectively. As Fig. 5 shows, we were able to obtain the two membrane preparations largely free of cross-contamination (compare lanes 1 and 7 with lanes 2 and 5). The isolated BM was heavily radiolabeled with [35S]sulfate (lanes 1 and 7), reflecting the synthesis of and presence in the BM of chondroitin sulfate, as demonstrated by the loss of label upon treatment of the isolated 35S-sulfated BM with chondroitinase ABC (lane 8 versus lane 7). The large 35S-labeled 210-kDa band in the isolated ZP was also chondroitinase ABC sensitive, as expected from its tentative identification as ovomucin (lanes 5 versus lane 6, and see Fig. 1).4 These results indicated that the macromolecular BM incorporated newly synthesized ggBM1. To determine whether soluble ggBM1 was present in the medium as well, aliquots of the medium were subjected to immunoprecipitation with anti-ggBM1 antibody (lanes 3 and 10) or preimmune serum as negative control (lanes 4 and 9). The specifically precipitated [35S]sulfate-labeled components were ggBM1 and a band at 84 kDa (lanes 3 and 4), likely representing one of the components observed above (Figs. 2 and 3). Immunoprecipitation of medium from [3H]proline-labeled GC resulted in several bands present in both the control and the anti-ggBM1 pellets (lanes 9 and 10); however, a broadly migrating radiolabeled band above the 116 kDa standard was only present in the immunoprecipitated material and had the typical appearance of ggBM1. Also, two bands at ~60 and 84 kDa, respectively, appeared to be more abundant in the anti-ggBM1 precipitate; the latter comigrated with the [35S]sulfate-labeled band (lane 3). Unfortunately, we did not obtain sufficient amounts of this material to analyze the effect of chondroitinase ABC treatment. The results from these experiments show that GC synthesize ggBM1 as component of the BM, in agreement with the basal localization of BM.


Figure 5
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  		FIGURE 5.
Analysis of radiolabeled biosynthetic products of granulosa cells. Isolated granulosa cell sheets from F2 follicles were incubated in Dulbecco's modified Eagle's medium containing Na235SO4 (lanes 1–8) or [2,3,4,5-3H]proline (lanes 9 and 10) as described under "Experimental Procedures." Subsequently the BM (lanes 1, 7, and 8) and the ZP (lanes 2, 5, and 6) were separated, and the media of the granulosa cell sheets were subjected to immunoprecipitation with anti-ggBM1 antiserum (lanes 3 and 10) or preimmune serum (lanes 4 and 9). Isolated ZP (lane 6) and BM (lane 8) from the Na235SO4 labeling experiment were treated with chondroitinase ABC. All samples were subjected to 8% SDS-PAGE and autoradiography. The position of ggBM1 is marked with an arrow. Molecular weight markers are indicated.

 
Yolk Precursors in the BM—Next, we tested whether based on the acidic properties of ggBM1 due to its chondroitin sulfate chains, the BM contained positively charged serum-derived yolk precursors such as VLDL particles that carry apolipoprotein B-100 (apoB) (30). To this end, we subjected extracts of isolated BM to immunoblotting with anti-apoB antiserum (Fig. 6, lane 1). Chicken apoB-100 is a protein with an Mr of ~520,000 (14) that is highly susceptible to proteolysis. For instance, following uptake into the yolk, apoB-100 is proteolyzed into smaller fragments, and the large apoB-100 completely disappears (14). However, the immunoreactive material in the BM clearly contained apo B-100 (lane 1), indicating that at least a large portion of the apoB was not derived from yolk contamination. Freshly isolated VLDL from laying hen serum showed a single immunoreactive band of approximately 520 kDa (lane 4). For control purposes, we also analyzed the isolated ZP (lane 2) and extracts of thecal tissue (lane 3), respectively. As expected, ZP lacked, and the theca extract contained, immunoreactive apoB fragments. In the theca, perlecan, which is absent from the BM is the protein mediating VLDL binding and storage (18) and is here confirmed by immunoblotting to be a thecal component (lane 5).


Figure 6
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  		FIGURE 6.
Analysis of apoB-100 in the follicular envelope. Follicular BM (lane 1), ZP (lane 2), theca (lanes 3 and 5), and plasma VLDL (lane 4) were isolated as described under "Experimental Procedures." Equal amounts (10 µg of protein/lane) were subjected to 4% SDS-PAGE under reducing conditions and subsequent Western blot analysis using the anti-apoB-100 anti-serum (lanes 1–4, 1:500 dilution) or anti-chicken perlecan antiserum (lane 5, 1:500 dilution). Horseradish peroxidase-conjugated goat anti-rabbit secondary antibodies were used at 1:50,000 dilution. Molecular weight markers are indicated.

 
Finally, we tested for the presence of the yolk precursor VLDL in situ by immunohistochemical analysis of follicle sections. Fig. 7 shows immunostaining of the BM with antibodies against the two proteins present on laying hen VLDL particles, i.e. apoB (A and C) and apo-VLDLII (B); in D, the ZP is immunostained with an antiserum raised against the major ZP protein, ZP1. The position of the BM is clearly distinct from that of the ZP, as can be seen by inspection of the position of granulosa cell nuclei relative to the immunostained structures (cf. A, B, and D). In summary, the results of Figs. 6 and 7 demonstrate that VLDL particles are concentrated in the BM, where ggBM1 is exclusively localized.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Several lines of evidence demonstrate that chicken ggBM1 is a collagen with covalently attached chondroitin sulfate chains synthesized by granulosa cells. First, the protein bound the dye Alcian blue, known to stain acidic glycoproteins well (Fig. 1); in fact, ggBM1 was the only Alcian blue-stainable protein in solubilized BM (Fig. 1, lane 4) and could be visualized with Alcian blue in follicle sections (Fig. 4). Second, ggBM1 was completely degraded by treatment with collagenase IV (Fig. 3). Third, protein sequences obtained from tryptic fragments of purified ggBM1 clearly identified multiple peptides with sequences typical for proteins of the large collagen family. Several results (Figs. 2, 3, and 5) suggest that ggBM1 might be closely related to collagen type II or IX. Fourth, in isolated BM preparations ggBM1 displayed migration in SDS-PAGE as a diffuse band centering around 135 kDa, which became a sharper 95-kDa band upon treatment with the chondroitin-specific hydrolase, chondroitinase ABC, but not, e.g., with the specific hyaluronidase from S. hyalurolyticus (Fig. 2). Fifth, strong evidence resulted from biosynthetic experiments performed with isolated granulosa cell monolayers (Fig. 5). The newly synthesized protein incorporated both radioabeled precursors, [3H]proline and [35S]sulfate, and was incorporated into the BM as well as secreted into the medium. Importantlly, the incorporated [35S]sulfate was completely removed by treatment of the biosynthetic product with chondroitinase ABC. As ggBM1 appears to have homologues in other avian species, e.g. in quail (Fig. 2, B and C), the role(s) of the chondroitin-sulfated collagen in follicles likely is important to the normal function of the BM. Experiments are now under way to further characterize the quail protein, and preliminary results are in support of a high degree of conservation of this component, at least in these two closely related avians.


Figure 7
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  		FIGURE 7.
Immunofluorescence images of chicken follicle sections with antisera against apo-yB5 (A), apo-VLDLII (B), apo-B100 (C), and ZP1 (D). Paraffin sections of chicken follicles (diameter, 4–5 mm) were incubated with the indicated rabbit antisera, followed by secondary antibodies as described under "Experimental Procedures." Immunoreactive proteins appear in green fluorescence; cell nuclei were counterstained with DAPI. Membranous structures are labeled with short solid lines when immunostained, and their position indicated with dashed lines when unstained. In A, yolk is intensely stained with the antiserum against the yolk-apoB fragment, yB5; in C, two stroma-embedded oocytes of approximately 50-µm diameter lacking a BM can be seen. TH, theca layers. The horizontal bars represent 100 µm.

 
In terms of functional aspects of ggBM1, the present studies provide support for an involvement in yolk precursor passage from the thecal microvasculature to the oocyte surface. First, negatively charged proteoglycans, notably heparin, heparansulfate, and chondroitin sulfate, are known to reversibly bind lipoproteins (31–35). Significantly, we found that Bruch's membrane of the chicken eye contains ggMB1 (Fig. 4C). This membrane has been shown to accumulate plasma lipids and lipoprotein components such as apolipoproteins, cholesterol, and triglycerides and may thereby contribute to the etiology of human age-related macular degeneration (28, 36). Second, the major yolk precursor VLDL is present in isolated BM (Fig. 6). High VLDL concentrations were revealed by intense immunostaining of the BM with two different antisera against apoB, as well as antiserum against apo-VLDLII, the other apolipoprotein of hen VLDL (Fig. 7). Previously, we have suggested that perlecan, a heparan sulfate proteoglycan in the thecal extracellular matrix, serves as the first attachment site for VLDL particles leaving the capillary bed (18). Indeed, here we confirmed perlecan to be present in the theca (Fig. 6), and apoB and apo-VLDLII were demonstrated in the theca (Figs. 6 and 7) but at lower local concentration than in the BM, likely due to the scattered distribution of the matrix harboring perlecan (18).

These findings are compatible with the hypothesis that the follicle component providing an intermediary holding site for VLDL particles subsequent to thecal perlecan is ggBM1 within the BM. Unfortunately, due to the poor reactivity and specificity of our anti-vitellogenin antibodies in Western blots and immunohistochemitry, we could not address the presence of vitellogenin, the other major yolk precursor (8), in the BM. However, the biochemical properties determining the interaction of vitellogenin with acidic molecules are highly reminiscent of apoB/VLDL (37), and thus it is very likely that serum vitellogenin follows the pathway of VLDL. Conceivably, ligand saturation of the intermediary storage sites is likely an advantage for the function of the oocyte-targeted delivery system. First, as the oocytes take up large amounts of VLDL and vitellogenin, low hepatic production levels of yolk precursors can be compensated for by their release from the stores in the theca and the BM. In turn, when production rates are high, the sites become saturated, and the precursors can pass through the matrices to the specific oocyte surface receptors that mediate their endocytosis (5). In fact, diurnal rhythm of production and thus access to the oocyte proper may explain the internal structure of yolk. Namely, yolk is deposited in seven concentric spherical shells, possibly corresponding to the 7 days of massive yolk deposition and oocyte growth (discussed in Ref. 18). An analogous situation involving ggBM1 might be the transport of macromolecular nutrients, including lipoproteins, to retinal cells. When serum components are released from the choriocapillaris (the primary capillary bed of the choroid), Bruch's membrane would provide the bridge to the retinal pigment epithelium cells. By analogy, these cells, like the granulosa cells for the oocyte (6), are support cells for the retina (28). Furthermore, both follicular BM and Bruch's membrane have been considered untypical BMs (18, 28) and are synthesized by and attached to the epitheloid cells of the respective organs.

The characterization of ggBM1 as chondroitin-sulfated collagen indicates several avenues for future investigations. First, despite circumstantial evidence for belonging to collagen families type II or IX, the gene specifying the ggBM1 polypeptide remains to be determined. With two sources (follicle and eye) now identified, chances for reaching this goal are improved. Second, while the lipoprotein receptors that function beyond ggBM1/BM in oocyte growth are known (6), those in the pigment epithelia have been identified only partially (38, 39). Currently, analysis of these cells for expression and presence of members of the LR gene family is under way in the laboratory. Third, although here we have referred to this interesting matrix (still) as BM, we anticipate that, with the molecular characterization of additional components, a novel type of membrane with dual function in both tissue compartmentalization and transport may become defined. The current studies underline our conclusion (18) that the follicular BM is not a bona fide BM, as already indicated previously by the observed absence of collagen IV, laminin B, perlecan, and fibronectin.


   FOOTNOTES
 
* This work was supported by research grants from the Austrian Science Foundation (FWF F-0608) and the Herzfelder Family Endowment. 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. Back

1 These authors contributed equally to this work. Back

2 To whom correspondence should be addressed: Dept. of Medical Biochemistry, Dr. Bohr Gasse 9/2, A-1030 Vienna, Austria. Tel.: 43-1-4277-61803; Fax: 43-1-4277-61804; E-mail: wolfgang.schneider{at}meduniwien.ac.at.

3 The abbreviations used are: LR, lipoprotein receptor; VLDL, very low density lipoprotein; BM, basement membrane; PBS, phosphate-buffered saline; ZP, zona pellucida; DAPI, 4',6-diamidino-2-phenylindole; HPLC, high pressure liquid chromatography; GC, granulosa cell(s). Back

4 A. Ducret, R. Aebersold, D. L. Barber, and W. J. Schneider, unpublished data. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Willi Halfter, University of Pittsburgh, for preparation of the chicken embryo eye section (Fig. 4).



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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