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Biology of Reproduction 59, 1334-1341 (1998)
©Copyright 1998 Society for the Study of Reproduction, Inc.

Distribution of the {alpha}1 to {alpha}6 Chains of Type IV Collagen in Bovine Follicles1

Helen F. Rodgersa, Charmaine M. Irvinea, Ingrid L. van Wezela, Tina C. Lavranosa, Martin R. Luckb, Yoshikazu Sadoc, Yoshifumi Ninomiyad, and Raymond J. Rodgers2,a

a Department of Medicine, Flinders University of South Australia, Bedford Park, South Australia 5042, Australia b Division of Animal Physiology, School of Biological Sciences, University of Nottingham, Sutton Bonington Campus, Loughborough LE12 5RD, United Kingdom c Division of Immunology, Shigei Medical Research Institute, Okayama, 701–0202, Japan d Department of Molecular Biology and Biochemistry, Okayama University Medical School, Okayama, 700–8558, Japan


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
During follicular development the proliferative and differentiated state of the epithelioid granulosa cells changes, and the movement of fluid across the follicular basal lamina enables the formation of an antrum. Type IV collagen is an important component of many basal laminae. Each molecule is composed of three {alpha} chains; however, six different type IV collagen chains have been identified. It is not known which of these chains are present in the follicular basal lamina and whether the type IV collagen composition of the basal lamina changes during follicular development. Therefore, we immunolocalized each of the six chains in bovine ovaries using antibodies directed to the nonconserved non-collagenous (NC) domains. Additionally, dissected follicles were digested with collagenase to release the NC domains, and the NC1 domains were then detected by standard Western immunoblot methods. The follicular basal lamina of almost all primordial and preantral follicles was positive for all type IV collagen {alpha} chains. Colocalization of type IV collagen and factor VIII-related antigen allowed for discrimination between the follicular and endothelial basal laminae. Type IV collagen {alpha}1, {alpha}2, {alpha}3, {alpha}4, and {alpha}5 chains were present within the follicular basal lamina of only a proportion of antral follicles (17 of 22, 20 of 21, 15 of 18, 14 of 28, and 12 of 23, respectively), and staining was less intense than in the preantral follicles. Staining for the {alpha}1 and {alpha}2 chains was diffusely distributed throughout the theca in regions not associated with recognized basal laminae. The specificity of this immunostaining for {alpha}1 and {alpha}2 chains of type IV collagen was confirmed by Western immunoblots. As well as being detected in the basal lamina of approximately half of the antral follicles examined, type IV collagen {alpha}4 also colocalized with 3ß-hydroxysteroid dehydrogenase-immunopositive cells in the theca interna. Type IV collagen {alpha}6 was detected in the basal lamina of only one of the 16 antral follicles examined. Thus, the follicular basal lamina changes in composition during follicular development, with immunostaining levels being reduced for all type IV collagen chains and immunoreactivity for type IV collagen {alpha}6 being lost as follicle size increases. Additionally, immunoreactivity for {alpha}1 and {alpha}2 appears in the extracellular matrix of the theca as it develops.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Development of the ovarian follicle is characterized by dramatic changes in the granulosa compartment. The number of granulosa cells doubles 21 times from the primordial follicle to the 18-mm preovulatory follicle [1]. There is also differentiation of the membrana granulosa cells, with marked differences occurring between cells at different stages of follicular development and relative to their position within the membrana granulosa [2]. The follicular basal lamina surrounds the oocyte-(antrum)-granulosa complex throughout follicular development—excluding capillaries, white blood cells, and nerve processes from the granulosa compartment—until it is degraded at ovulation. It has been postulated that the follicular basal lamina regulates the fate of the granulosa cells [3], consistent with the role of basal laminae in influencing cell proliferation, differentiation, and migration and in maintaining polarity of other cell types. The follicular basal lamina might also play a role in filtering out the larger molecules of serum during the accumulation of follicular fluid [4].

The functions of basal laminae throughout the body are very tightly related to their composition. Basal laminae are often composed of a lattice-type network of type IV collagen intertwined with a network of laminin. This structure is stabilized by the binding of entactin to the collagen and laminin and by low-affinity interactions between type IV collagen and laminin [5, 6]. Fibronectin, heparan sulfate proteoglycans, and other molecules are associated with the type IV collagen-laminin backbone. Importantly, basal laminae in different regions of the body differ in the ratio of all these components. Furthermore, each "component" is a class of several isoforms. The type IV collagen molecule is composed of three separate {alpha} chains; but six different isoforms, encoded by separate genes, have been discovered to date. Potentially, any combination of the chains might be present, although some combinations such as ([{alpha}1]2, {alpha}2), ({alpha}3, {alpha}4, {alpha}5), and ({alpha}5 with {alpha}6) are more common than others [7–9]. It is considered that the unique composition of each basal lamina, determined by the ratios of the different components to each other and of the specific isoforms of each component present, contributes to its specific functional properties [10]. For example, changes in the composition of a basal lamina can affect its ability to selectively filter materials [11], as occurs in the renal glomerulus.

During follicular development, there is a 317 400-fold increase in the area covered by the basal lamina from the primordial follicle to the 18-mm follicle [1]. This implies that the follicular basal lamina is continually remodeled during follicle growth. We have previously hypothesized that the composition of the follicular basal lamina changes during follicular development and atresia [12]. Consistent with this, the presence of individual chains of laminin was shown to alter with follicular development and atresia. Immunolocalization studies of the follicular basal lamina have demonstrated the presence of type IV collagen [13–15] but have not identified which isoforms are present. In another study utilizing Northern blotting, the expression of the {alpha}2 chain was detected in granulosa cells, and the expression of the {alpha}3 chain was detected in both the theca and granulosa cells [16]. The chains were not specifically localized to the follicular basal lamina; this is important, as there are other basal laminae in follicles, such as those of the thecal vasculature, and our studies have identified an extracellular "thecal matrix" [12]. Furthermore, none of these studies have considered changes in the collagen component of the follicular basal lamina with follicular development and atresia.

As type IV collagen is one of the most important structural components of basal laminae, in the current work we investigated the specific type IV collagen composition of the follicular basal lamina and the ways in which it changes with follicular development. Type IV collagen was immunolocalized in bovine follicles, and Western blot analyses were conducted on isolated follicles using antibodies specific to the {alpha}1, {alpha}2, {alpha}3, {alpha}4, {alpha}5, and {alpha}6 chains of type IV collagen.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Tissues

All bovine tissue was collected at a local abattoir, within 20 min of slaughter, from cows assessed visually as being nonpregnant. A slice of up to 5 mm was cut through the center of ovaries (n = 10) to be used for immunohistochemistry, and the slices were immediately immersed in Tissue-Tek OCT embedding compound (Miles Inc., Elkhart, IN) and snap-frozen; these blocks were stored at -70°C. Tissue sections (10 µm) were cut using a CM1800 Leica (Milton Keynes, Bucks, UK) cryostat, collected on glass slides freshly treated with 0.01% poly-L-lysine hydrobromide (cat. #P-1524; Sigma Chemical Co., St. Louis, MO) or 0.01% poly L-ornithine hydrobromide (cat. #P-4638; Sigma), and stored at -20°C until use. For Western blot analyses, tissues were placed into Hepes-buffered Earle's balanced salt solution without calcium or magnesium and placed on ice during transport to the laboratory. Antral follicles (3–10 mm) were dissected from the ovaries, and all adhering surface epithelium was removed. Follicles were pooled on the basis of diameter into four categories: < 3 mm (n = 10 follicles); 3–5 mm (n = 9 follicles); 5–10 mm (n = 7 follicles); > 10 mm (n = 4 follicles). The follicular fluid was gently removed by aspiration, and the remaining tissue of each follicle was weighed (pooled weights = 30 mg, 109 mg, 304 mg, 527 mg for categories 1–4, respectively) and stored at -20°C until required. Pieces of two kidneys were similarly weighed and stored.

Antibodies

The current study used both rat monoclonal antibodies and mouse monoclonal antibodies directed against individual {alpha} chains of type IV collagen. The rat monoclonal antibodies were to each of the type IV collagen {alpha}1 to {alpha}6 chains, and they were originally raised against synthetic peptides derived from amino acid sequences of the human non-collagenous (NC) 1 domain of each chain [17]. These antibodies have been screened by ELISA with a synthetic peptide and native NC1 fractions from human, rat, or bovine renal basal laminae and further screened by indirect immunohistochemistry of human kidney. They have also been shown previously to cross-react well with bovine tissue with the exception of the type IV collagen {alpha}6 antibody, which was less reactive (unpublished results). The mouse monoclonal antibodies were to the {alpha}1, {alpha}3, and {alpha}5 chains (Wieslab AB, Lund, Sweden). The type IV collagen {alpha}1 and {alpha}3 antibodies were raised against purified bovine NC1 domains; they were characterized by immunohistochemistry on human kidney sections and by Western blot analyses and ELISA against denatured and native NC1 hexamers from human and bovine kidney, human placenta, and bovine lens capsule [18]. The {alpha}5 antibody was raised against the collagenase-resistant residue of human glomerulus basement membrane. It was screened by ELISA and SDS-PAGE to recombinant human type IV collagen chains and further characterized by indirect immunofluorescence within human kidney sections [19, 20]. This detects the bovine {alpha}5 chain of type IV collagen (J. Wieslander, personal communication). All of these specific primary antibodies were diluted to 1:100 in antibody diluent (291 mM NaCl, 7.54 mM Na2HPO4, 2.50 mM NaH2PO4·2H2O, 0.01% NaN3, pH 7.1). The rat monoclonal antibodies were used under denaturing and nondenaturing conditions, whereas the mouse monoclonal antibodies were used only under nondenaturing conditions.

Rabbit anti-human von Willebrand factor (factor VIII-related antigen) was obtained commercially (cat. #F-3520; Sigma, 1:50 in antibody diluent). Rabbit antiserum to 3ß-hydroxysteroid dehydrogenase (3ß-HSD) type II was kindly donated by J.I. Mason (Clinical Biochemistry, University of Edinburgh; 1:50 in antibody diluent); this antiserum was raised against human placental type I 3ß-HSD [21, 22], but it cross-reacts with type II 3ß-HSD [21–23], as type I 3ß-HSD is 95% homologous to the type II 3ß-HSD that is expressed in gonads and the adrenal. Normal sera used in this study for control sections were normal rabbit serum (cat. #R9133; Sigma), normal mouse serum (cat. #M5905; Sigma), normal rat serum (cat. #R9759; Sigma), and normal donkey serum (cat. #D9663; Sigma).

Fluorophore-conjugated secondary antibodies used in this study were from Jackson ImmunoResearch Laboratories (West Grove, PA) and included Cy3-conjugated AffiniPure donkey anti-rat IgG (cat. #712–165–153), Cy3-conjugated AffiniPure donkey anti-mouse IgG (cat. #715–165–150), and fluorescein (DTAF)-conjugated AffiniPure donkey anti-rabbit IgG (cat. #711–095–152), each at a dilution of 1:100 in antibody diluent. For further amplification, secondary antisera used were biotin-SP-conjugated AffiniPure F (ab')2 fragment donkey anti-mouse IgG (H&L) (cat. #715–066–151) or biotin-SP-conjugated AffiniPure F (ab')2 fragment donkey anti-rabbit IgG (cat. #711–066–152), in conjunction with Cy3-conjugated streptavidin (cat. #016–160–084) or fluorescein (FITC)-conjugated streptavidin (cat. #016–090–084).

Immunohistochemistry

Cryosections were dried at room temperature under vacuum (30 min) prior to fixation in acetone (5 min). After washing in hypertonic PBS solution (hPBS; 274 mM NaCl, 5.37 mM KCl, 8.10 mM Na2HPO4, 1.47 mM KH2PO4, pH 7.2; three times, 5 min each), some sections were denatured with acidified urea solution (6 M urea, 0.1 M glycine, 0.1 M HCl, pH 3.5) [24] for 30 min and then washed again (three times, 5 min each in hPBS). The denaturation step was omitted when the mouse monoclonal antibodies were used. Nonspecific staining was inhibited by the preincubation of sections with 10% normal donkey serum in hPBS (30 min). Sections were incubated overnight with either specific primary antiserum or nonimmune control serum. Sections were then washed (four times, 5 min each in hPBS) and incubated with the appropriate fluorophore-conjugated secondary antibodies for 2 h. To achieve further amplification of the immunostaining, in some instances sections were instead incubated with secondary antisera conjugated with biotin-SP (diluted with antibody diluent) for 2 h; they were then washed (four times, 10 min each in hPBS) and further incubated with Cy3- or FITC-conjugated streptavidin (1:100 diluted in antibody diluent) for 1 h. All incubations were carried out at room temperature in a humidified environment. After final washing (four times, 10 min each in hPBS), sections were mounted with buffered glycerol (0.167 M Na2CO3 in 67% glycerol, pH 8.6).

The protocol for dual labeling was essentially the same as for single labeling as described above, except that the sections were incubated concurrently with two primary antibodies of different species and were subsequently incubated concurrently with the two appropriate secondary antisera to enable discrimination between the primary antibodies. These were conjugated either with different fluorophores or with one conjugated to biotin-SP to allow further amplification by incubation with streptavidin conjugated to either Cy3 or FITC. Control sections for dual staining were as follows: the relevant polyclonal primary antibody and anti-mouse secondary antibody; the relevant mouse monoclonal primary antibody and anti-rabbit secondary antibody; the relevant rat monoclonal primary antibody and anti-rabbit secondary antibody; normal rabbit serum primary antibody and anti-rabbit secondary antibody; normal mouse serum primary antibody and anti-mouse secondary antibody; normal rat serum primary antibody and anti-rat secondary antibody; both normal rabbit serum and normal mouse serum primary antibodies and both anti-rabbit and anti-mouse secondary antibodies; both normal rabbit serum and normal rat serum primary antibodies and both anti-rabbit and anti-rat secondary antibodies.

Observations and Photography

Immunostaining was visualized using either an Olympus (Tokyo, Japan) AX70 fluorescent microscope, with the selective NG filter (575–615 emission) for detecting the Cy3 fluorophore and an NIB filter (515–545 emission) for DTAF fluorophore, or an Olympus Vanox AHBT3 fluorescence microscope using the IB filter (490 emission) to excite the DTAF fluorophore and the G filter (546 emission) to excite the Cy3 fluorophore. Images were captured using the AX70 microscope via a video linkage to an Apple Macintosh (Cupertino, CA) computer utilizing the program NIH Image 1.60b7 or were photographed using the Vanox microscope with Olympus C35AD-4 camera attachment. Photographs used in all the illustrations here were taken using Kodak (Eastman Kodak, Rochester, NY) T-Max 400 black-and-white film.

Western Blotting of Type IV Collagen NC1 Domains

To release the NC1 domain of type IV collagen, the tissues were homogenized and then digested at 37°C for 24 h with collagenase (50 mg/ml, CLS1, 238 U/mg; Worthington Biochemical Corporation, Freehold, NJ) in Hepes-buffered (50 mM; pH 7.5) calcium chloride solution (10 mM) containing protease inhibitors (4 mM N-ethylmalemide, 1 mM PMSF, 5 mM benzamidine hydrochloride, 25 mM {epsilon}-aminocaproic acid [25–27]); 2 ml of collagenase solution was used per gram of tissue. The digestion was terminated by the addition of EDTA (to a final concentration of 25 mM). Samples were then centrifuged (10000 x g; 10 min), and the supernatants were stored at -20°C until required.

Samples were denatured by the addition of an equal volume of 0.05 M Tris buffer, pH 6.8, containing 2% SDS, 10% glycerol, 2 mM EDTA, 0.005% bromophenol blue, 0.1 M 4,4'diaminodiphenyl sulfone, 12 M urea solution, at 100°C for 5 min. The samples were subsequently electrophoresed on 12.5% SDS-polyacrylamide gels, and the separated proteins were electroblotted onto nitrocellulose-coated nylon membrane (Hybond-C; Amersham, Castle Hill, NSW, Australia) at 175 mA overnight (transfer buffer: 20% methanol, 20 mM Tris, 150 mM glycine). A lane containing molecular weight markers (cat. #17–0446–01; Pharmacia Biotech, North Ryde, NSW, Australia) was removed and stained with Napthol blue black, and the remaining lanes were developed in a manner adapted from Towbin et al. [28] as reported previously [29]. Nonspecific binding sites were blocked by incubating the membrane (buffer A: 10 mM Tris pH 7.4, 150 mM NaCl, 5% BSA, 0.2% Nonidet P-40) for 1 h at 37°C in a shaking incubator. Each membrane was subsequently incubated with one of the antibodies directed against a type IV collagen {alpha} chain diluted 1:2500 in buffer A for 2.5 h at room temperature. Each blot was then washed (three times, 15 min each) in buffer A without BSA, but with the addition of 0.25% deoxycholic acid and 0.1% SDS, followed by a further rinse (once, 10 min) in a solution of 10 mM Tris and 0.15 M NaCl. The appropriate secondary antisera (goat anti-rat IgG; cat. #R5005; Sigma) and goat anti-mouse IgG (cat. #M8645; Sigma) were iodinated using the lactoperoxidase method as described previously [29, 30] and were diluted to 1 x 106 cpm/ml in buffer A. Blots were incubated with the secondary antisera for 45 min at room temperature and then washed as described above and air dried. Blots were subjected to autoradiography (Kodak XAR film) or analyzed using a Molecular Dynamics (Sunnyvale, CA) PhosphoImager and ImageQuant (Molecular Dynamics) software, version 4.1.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
There are many structures in the ovary that contain basal laminae, but comments in this paper will be confined to ovarian follicles and their basal laminae. Specific staining for each of the individual type IV collagen chains was observed in basal laminae associated with ovarian follicles (summarized in Table 1; described in more detail below). All illustrations in this paper are of staining produced using the rat monoclonal antibodies unless otherwise specified. Only one antibody was available for type IV collagen {alpha}6, and this antibody cross-reacted with bovine {alpha}6 only weakly, as shown previously (Y. Sado, unpublished results). Dual labeling was also carried out to identify cell types such as endothelial cells (positive for factor VIII-related antigen) and steroidogenic thecal cells (positive for 3ß-HSD). A comprehensive series of controls was carried out to ensure that no nonspecific staining or autofluorescence was misinterpreted as positive staining.


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  		Table 1. Summary staining patterns produced by antibodies specific to the individual type IV collagen chains.*

Follicular Basal Laminae of Primordial and Preantral Follicles

The follicular basal laminae of almost all primordial and small preantral follicles were positive for type IV collagen {alpha}1, {alpha}2, {alpha}3, {alpha}4, and {alpha}5 (Fig. 1) and stained weakly for {alpha}6 (not shown). Only a very few primordial follicles did not appear to contain detectable {alpha}3, {alpha}4, {alpha}5, or {alpha}6 in their follicular basal laminae. No staining with antibodies to factor VIII-related antigen was observed in the region of the ovary containing the primordial follicles, confirming that this region of the ovary is avascular [1].



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  		FIG. 1. Immunofluorescent localization of individual {alpha} chains of type IV collagen to preantral follicles (including primordial follicles). Primary antibodies used were directed against: a) the {alpha}1 chain, b) the {alpha}2 chain, c, d) the {alpha}3 chain (mouse monoclonal), e–g) the {alpha}4 chain, h, i) the {alpha}5 chain. Arrows indicate the position of the follicular basal laminae. Bar (lower right in i) = 50 µm in a–e, g, h; 100 µm in f, i.

Type IV Collagen {alpha}1 and {alpha}2 in Antral Follicles

Antibodies to the {alpha}1 and {alpha}2 chains of type IV collagen in antral follicles produced similar staining patterns (Fig. 2). In all of the follicles examined for {alpha}1(IV) staining and all except one of the antral follicles examined for {alpha}2(IV) staining, intense uneven staining was observed extracellularly throughout the theca interna (Fig. 2, a–c and e–g). Its close proximity to the follicular basal lamina made staining of the follicular basal lamina difficult to interpret. Of the 22 follicles examined for {alpha}1(IV) staining, 17 were considered to have basal laminae that stained positively; of these, 14 stained with intensity equal to that for the thecal matrix, 2 stained more intensely, and 1 stained less intensely. In 5 of the 22 follicles examined for {alpha}1(IV), we were not confident in classifying the basal lamina as staining positively; if there was staining, it was at the same intensity as the thecal staining. Of the 21 follicles examined for {alpha}2(IV) staining, 20 were considered to have basal laminae that stained positively, and 1 did not stain for this chain. Of the basal laminae that did stain, 18 stained with intensity equal to that in the theca, although staining was often uneven in intensity along the basal lamina. The basal lamina of two follicles stained more intensely than the theca.



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  		FIG. 2. Immunofluorescent localization of type IV collagen {alpha}1 and {alpha}2 chains to antral follicles, and colocalization of factor VIII-related antigen. In each photograph, the location of the basal lamina is indicated by arrows, the membrana granulosa lies above the basal lamina and the theca below it, and the follicular antrum is uppermost. a–c) Positive staining for type IV collagen {alpha}1 in the follicular basal lamina as well as widespread staining in the theca are shown. Counterstaining of the section shown in c for factor VIII-related antigen, shown in d, demonstrated that the type IV collagen {alpha}1 staining is more widespread than the thecal vasculature. Similarly, staining for type IV collagen {alpha}2 (e–g) is present in the follicular basal lamina and widespread in the theca. Counterstaining of the section shown in g for factor VIII-related antigen, shown in h, demonstrated that the type IV collagen {alpha}2 staining is more widespread than the thecal vasculature. Follicle sizes: a, 3 mm; b, f (same follicle), 10 mm; c, d (same follicle), 5 mm; e, 9 mm; g, h (same follicle), 8 mm. Arrowheads indicate the vasculature. Bar = 50 µm.

Some of the extracellular staining observed in the theca interna was clearly associated with the endothelial basal laminae, but there were more areas staining positively for the {alpha}1 and {alpha}2 chains of collagen type IV than could be accounted for by the vasculature alone (compare Fig. 2, c with d and g with h).

Type IV Collagen {alpha}3, {alpha}4, {alpha}5, and {alpha}6 in Antral Follicles

Although the follicular basal laminae of some antral follicles stained for individual collagen chains (Fig. 3), this was not the case for all follicles; and the intensity of staining was much reduced compared to that of primordial and small preantral follicles. Staining was present in the follicular basal lamina of 15 of 18 follicles examined for {alpha}3 staining but was discontinuous along the basal lamina and/or punctate. The follicular basal laminae of only 14 of 28 follicles examined for {alpha}4(IV) staining were positive. However, staining for {alpha}4(IV) was faintly present intracellularly in cells in the theca interna (Fig. 3g) of all except 2 follicles. Colocalization studies with anti-3ß-HSD (Fig. 3h) showed that the cells positive for {alpha}4(IV) also stained for 3ß-HSD. The follicular basal lamina of 12 of 23 follicles examined stained positively for {alpha}5(IV); the remainder had no detectable staining. Of 16 follicles examined, 15 contained no detectable {alpha}6(IV) in their follicular basal laminae, and staining was punctate in the follicular basal lamina of the other follicle.



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  		FIG. 3. Immunofluorescent localization of individual type IV collagen {alpha} chains to antral follicles, and colocalization of type IV collagen {alpha}4 with 3ß-HSD. In each photograph, the location of the follicular basal lamina is indicated by arrows, the membrana granulosa lies above the basal lamina and the theca below it, and the follicular antrum is uppermost. a–f) Show positive staining for type IV collagen {alpha} chains in the follicular basal lamina. a, c, e) Healthy follicles; b, d, f) all are of the same atretic follicle. g) Staining of the theca, using the primary antibodies directed against type IV collagen {alpha}4; h) the same section counterstained for 3ß-HSD. Arrowheads indicate the vasculature. Bar = 50 µm.

Immunostaining of the Thecal Vasculature

The endothelial cell marker, factor VIII-related antigen, was colocalized with each of the type IV collagen {alpha} chains. In antral follicles, the arterioles and venules were located mainly at the extremities of the theca interna and in the externa, and capillaries were common in the theca interna. Intense fluorescence was observed in the subendothelial basal lamina of arterioles, venules, and capillaries with antibodies to {alpha}1 and {alpha}2, and the basal laminae of smooth muscle cells of arterioles also contained {alpha}1 and {alpha}2. In the thecal layers, no fluorescence was observed in the subendothelial cell basal laminae or the basal laminae of the vascular smooth muscle cells using antibodies to {alpha}3, {alpha}4, {alpha}5, or {alpha}6.

Western Blotting

In basal laminae, type IV collagen molecules assemble into a meshwork via disulfide bonds and by other nonreducible covalent cross-linking bonds [31]; therefore we digested the collagenous domains of type IV collagen with collagenase, releasing the NC1 domains. Each NC1 domain is a trimer of three chains, and each NC1 domain is bonded to the NC1 domain of another molecule of type IV collagen. Upon reduction, these hexameric structures yield monomers (approximately 25 kDa) and dimers (approximately 50–60 kDa) of single peptide chains from the NC1 domains [31]. The dimers are due to nonreducible, covalent cross-links [31]. Bovine kidney was chosen as a positive control as this tissue had previously been reported to contain four type IV collagen chains: {alpha}1, {alpha}2, {alpha}3, and {alpha}4 [27].

All of the rat monoclonal antibodies to type IV collagen {alpha}1 to {alpha}5 chains reacted well with bovine kidney (shown only for {alpha}1 and {alpha}2; Fig. 4). The mouse monoclonal antibodies were not tested. Strong specific bands of approximately equal intensity were observed at 54 and 28 kDa for both {alpha}1(IV) and {alpha}2(IV). Staining for {alpha}3(IV) (53 kDa and a stronger band at 28 kDa), {alpha}4(IV) (54, 28, and 25 kDa), and {alpha}5(IV) (54 kDa and a strong band at 28 kDa) was observed. The type IV collagen {alpha}6(IV) antibody barely detected any positive bands. The presence of two monomers detected using type IV collagen {alpha}4 antibody is likely to be due to interchain, nonreducible, covalent cross-links as has been observed previously [25]. The rat monoclonal antibodies to the {alpha}1 to {alpha}5 chains also reacted with nonreduced NC1 domains (not shown) but more strongly with denatured products.



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  		FIG. 4. Western blot analyses of type IV collagen {alpha}1 and {alpha}2. Samples were digested with collagenase, and monomers (small arrowhead) and dimers (large arrowhead) were detected. Lane 1, bovine kidney; lane 2, follicles < 3 mm; lane 3, follicles 3–5 mm; lane 4, follicles 5–10 mm; lane 5, follicles > 10 mm.

Western blot analyses of dissected antral follicles showed that {alpha}1(IV) and {alpha}2(IV) were readily detectable at levels similar to that in kidney (Fig. 4). In comparison, the levels of the other type IV collagen chains {alpha}3, {alpha}4, or {alpha}5 were far lower in the follicles. This is consistent with the immunostaining patterns observed; by immunostaining, a high level of staining for {alpha}1 and {alpha}2 was present throughout the theca, whereas the {alpha}3 and {alpha}5 chains were observed only in the follicular basal laminae, and the thecal intracellular staining for {alpha}4 was very weak.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This is the first study to localize any of the six {alpha} chains of type IV collagen within the follicular basal lamina of any species. The follicular basal lamina of primordial and small preantral follicles contained all of the {alpha} chains of type IV collagen. In antral follicles, there was widespread extracellular staining throughout the theca interna for the {alpha}1 and {alpha}2 chains, often making it impossible to determine whether the follicular basal lamina itself contained these chains. The {alpha}3, {alpha}4, and {alpha}5 chains were not detected in some antral follicles. When it was present, the level of staining was less intense than at earlier stages of follicular development. The {alpha}6 chain was not detected in the follicular basal lamina of any antral follicles. In the light of the current study and a previous study of individual laminin chains in the ovarian follicle [12], it is clear that important changes take place during follicular development, both in the composition of the follicular basal lamina and in the development of a thecal matrix in the mesenchyme, adjacent to the follicular basal lamina.

There are currently 15 different classes of collagen; however, only type IV collagen is found predominantly in basal laminae. Each type IV collagen molecule contains three {alpha} chains wound into a triple helix. In all, six different {alpha} chains have been discovered, and a type IV collagen molecule may contain any combination of these chains although some combinations, such as {alpha}1(IV)2 {alpha}2(IV), are more common than others [8]. Here, we have demonstrated for the first time that the presence of individual type IV collagen {alpha} chains in the follicular basal lamina alters with follicular development. This is consistent with studies in other developing tissues. For example, during development of the kidney, collagen {alpha}1(IV) and {alpha}2(IV) are lost from the glomerular basement membrane, whereas collagen {alpha}3(IV), {alpha}4(IV), and {alpha}5(IV) are accumulated [11]. Additionally, studies of the type IV collagen {alpha}1 to {alpha}5 chains in murine seminiferous tubules have shown a temporal expression pattern of these chains during tubule development [32, 33]. Similar changes in individual chains of laminin within basal laminae of developing tissues are correlated with functional changes ([11]; see [12]). Therefore it is likely that the changing expression patterns reflect specific functional roles for individual type IV collagen chains. Such functional roles, though, have yet to be determined for any of these chains. Additionally, it remains to be seen which {alpha} chains combine to form type IV collagen molecules, how the type IV collagen molecules are combined in the follicular basal lamina (see [9]), and whether different arrangements of the molecules affect the function of the follicular basal lamina.

In the current study, follicular development was associated with a decrease in intensity or a complete loss of type IV collagen chains from the follicular basal lamina. Additionally, staining for {alpha}1(IV) and {alpha}2(IV) was frequently unevenly distributed along the follicular basal lamina of antral follicles, being present in some areas and absent in others. Conversely, in our previous study of laminin chains, the intensity of staining increased with increasing follicle size [12]. Thus there appears to be a transition from a type IV collagen-rich to a laminin-rich follicular basal lamina with increasing follicle size. Type IV collagen molecules can be covalently cross-linked to form a much more rigid network than the laminin network, and absence or discontinuity of the type IV collagen network might reflect the need for active basal lamina expansion during follicular development. It has been calculated that the surface area of the bovine follicle, the area covered by the follicular basal lamina, increases some 317 400-fold or doubles 19 times in developing from the primordial follicle stage to the 18-mm preovulatory-sized follicle [1]. Thus there must be continued synthesis and substantial remodeling of the follicular basal lamina. Incorporation of a new segment into the follicular basal lamina would require that this network be broken, and segments of the basal lamina lacking type IV collagen would easily be able to expand by incorporating new components. Follicles that did not contain any of the type IV collagen {alpha} chains might be those that are actively growing and require rapid basal lamina expansion, as a basal lamina containing no type IV collagen would be easily remodeled. Basal laminae lacking type IV collagen have been found in a range of tissues during their development [34–36] and in a basal lamina produced in vitro [37].

The ovarian cell type that is responsible for producing the components of the follicular basal lamina remains controversial, although there is mounting evidence to suggest that the granulosa cells make a substantial, if not the sole, contribution to its synthesis. In other systems it is predominantly epithelial rather than stromal cells that synthesize basal lamina components, although in some tissues both cell types make a contribution [38]. In the ovarian follicle, it is the granulosa cells that are present throughout follicular development associated with the follicular basal lamina. The theca, on the other hand, only differentiates in bovine follicles at approximately the time of antrum formation, well after the basal lamina of the follicle has started increasing. Cultured granulosa cells have been shown to produce a basal lamina [39] that structurally resembles the follicular basal lamina [40]. By Northern blot analysis, granulosa cells were shown to express the {alpha}3 chain of type IV collagen and the {gamma}1 (old nomenclature: B2) chain of laminin [16]. In a previous immunoelectron microscopy study [41], laminin was localized to Call-Exner bodies, which are ultrastructurally similar to basal lamina and have been observed within the membrana granulosa of antral follicles in vivo (cow: unpublished results; rabbit: [42]). Conversely, in some systems, mesenchymal production of basal lamina components have been shown [43], suggesting that cells of the theca might play a role in production of the follicular basal lamina. The thecal compartment of antral follicles has been shown by Northern analyses to express the {alpha}2 and {alpha}3 chains of type IV collagen and the laminin ß1 and {gamma}1 chains [16], and our observations of the {alpha}1 and {alpha}2 chains of type IV collagen (current study) and laminin {gamma}1 [12] in the `thecal matrix' suggest that cells in the thecal layer are capable of producing basal lamina components. However, the thecal expression of these molecules is not necessarily a contribution to the follicular basal lamina. This was particularly demonstrated for the laminin ß1 chain, which was immunolocalized to the thecal vasculature but undetectable in the follicular basal lamina at most stages of follicular development [12]. Nevertheless, it remains possible that in vivo, the follicular basal lamina receives a contribution from both the granulosa cells and the stroma or thecal cells.

Type IV collagen has been reported to be found in basal laminae. However, in the current immunolocalization study, relatively large amounts of {alpha}1(IV) and {alpha}2(IV) were found within the extracellular regions of the theca interna of antral follicles. These large diffuse areas of staining were not associated with the follicular basal lamina or the subendothelial or smooth muscle basal laminae of the vasculature. The staining was not likely to be nonspecific, as Western immunoblot analyses of dissected follicles using the same antibodies produced bands only at the expected molecular weights for type IV collagen {alpha} chains, namely NC1 dimers (54 kDa) and monomers (28 kDa). Furthermore, staining patterns similar to those observed in the current study were reported by Bagavandoss et al. [13] using an antibody to the EHS sarcoma type IV collagen; {alpha}1(IV) and {alpha}2(IV) are found in the EHS sarcoma. Widespread staining in the theca interna has also been observed for laminin using antibodies directed against either the EHS laminin [12, 13, 41, 42], which contains the {alpha}1, ß2, and {gamma}1 laminin chains, or against the {gamma}1 chain alone [12]. It is possible that the collagen {alpha}1(IV) and {alpha}2(IV) chains and the laminin {gamma}1 chain are present as free molecules in the theca interna, as there are no recognized conventional basal laminae in these regions. However, it is also possible that this staining is associated with small fragments of basal lamina-like electron-dense material found extracellularly in the theca. This material has been observed at the electron microscope level in the theca of sheep [44], rats [41], and cows (see Fig. 1 in [29]). The origins and functions of this thecal matrix are not known.

From our studies, we conclude that the composition of the follicular basal lamina changes during the course of follicle growth and development. We believe, but have still to prove, that these changes reflect changes in the function of the follicular basal lamina, particularly in filtration properties and their ability to regulate functions of granulosa cells. We have also to determine for type IV collagen (and also laminin) how the individual chains are combined in each molecule and whether this also changes with follicle development. The large amounts of {alpha}1(IV) and {alpha}2(IV) present in the matrix of the thecal layer of the follicle are not associated with the follicular basal lamina or the basal laminae of the vasculature. The role of this matrix in the differentiation of thecal cells or other functions has still to be determined.


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

2 Correspondence. FAX: 61 8 82045450; ray.rodgers{at}Flinders.edu.au Back

Accepted: July 21, 1998.

Received: May 28, 1998.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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