ARTICLE |
Correspondence to: Hiroshi Kobayashi, Dept. of Obstetrics and Gynecology, Hamamatsu University School of Medicine, Handacho 3600, Hamamatsu, Shizuoka, 431-3192, Japan. Fax: +81 53 435 1626.
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Summary |
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To gain insight into the role of link protein in ovarian follicle development, we used immunohistochemistry to determine the patterns of link protein expression in mouse ovary in response to gonadotropin stimulation. Polyclonal antibodies were raised against link protein purified from bovine cartilage. Stimulation of immature mice with gonadotropins increased link protein expression in the granulosa layer of large preovulatory follicles. The number and intensity of immunostained cells increased over 2 hr after hCG injection. Cumulus cells stained link protein mainly in the extracellular matrix, whereas mural granulosa cells showed marked deposits of link protein in the cytoplasm. Link protein expression persisted in luteinized granulosa cells after ovulation and in corpora lutea. Link protein staining was also present in the theca cells and oocytes, which was a consistent finding regardless of gonadotropin treatment. The staining intensity was negated by treatment with hyaluronidase, suggesting that the link protein is bound to hyaluronic acid. On Western blotting, a reacting protein species of about 42 kD was seen in the gonadotropin-treated ovarian extract. The precise cellular distribution of link protein in mouse ovary was determined for the first time by an immunohistochemical method in this study. (J Histochem Cytochem 47:14331442, 1999)
Key Words:
cumulus oocyte complex, expansion, hyaluronic acid, inter--trypsin inhibitor, link protein
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Introduction |
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The development of ovarian follicles is considered to be regulated by various factors such as gonadotropins (-trypsin inhibitor (I
I) (
I family in the gonadotropin-stimulated cumulus cells (
It has been reported that the dermatan sulfate proteoglycan and the ~46-kD protein synthesized by the cumulus cells form ternary complexes that are necessary for retaining HA in the COC matrix (I (
I or other extracellular matrix proteins and may thus influence the spacing of the monomers along the HA filament during follicle development.
There is no direct evidence that the ~46-kD protein reported by
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Materials and Methods |
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Preparation of Link Protein
The isolation of HA binding protein derived from bovine nasal cartilage has been described in detail elsewhere (
Twenty mg of HA binding protein was concentrated to a volume of 2 ml using a Centricon 10 ultrafiltration tube by centrifugation at 200 x g for 15 min at 4C and was then further purified by gel filtration chromatography on a column of Sepharose CL-6B (2.5 x 175 cm) equilibrated in 4 M guanidine-HCl, 50 mM Tris-HCl, pH 7.4, as described by
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Preparations of Polyclonal Antibodies Against Bovine Link Protein and Its Synthetic Peptides
Polyclonal antibodies against bovine nasal cartilage link protein were prepared by intradermal injection of rabbits with 0.2 mg of purified link protein emulsified in Freund's complete adjuvant. Four weeks after the first injection, the rabbit was boosted with 0.1 mg of protein in incomplete Freund's adjuvant, and then boosted again at 4-week intervals. This antiserum had a 50% maximal binding at a dilution of 1:10,000 in a specific ELISA with link protein used for immmunization as antigen. This antiserum was reactive with both 42-kD (ovary) and 49-kD and 40-kD (cartilage) proteins in Western blot assay. This antiserum reacts with purified link proteins but not with aggrecanHA binding region (unpublished data; and
Affinity-purified IgG was prepared by mixing 3 ml of antiserum with 1 ml of link protein-coupled Sepharose 4B overnight at 4C. After washing, the IgG was eluted with 100 mM glycine-HCl, pH 2.5. The pH of the eluted fractions was immediately raised and the IgG was stored at -20C. Polyclonal antibodies raised against bovine cartilage link protein reacted under nonreducing and reducing conditions in immunoblot analyses with purified bovine link protein and also with mouse link protein expressed in ovary. Interestingly, this antibody reacted with bovine and mouse link protein in immunoblot analyses and also in cryostat sections and in formalin-fixed, paraffin-embedded sections (see Results).
In addition, to generate anti-bovine link protein peptide antibodies, two synthetic oligopeptide sequences, 112VFLKGGSDNDAS123 and 231TVPGVRNYGFWDKDKS246, corresponding to the NH2-terminal domain and the COOH-terminal domain of bovine link protein molecule (GenBank Accession No. U02292), respectively, were selected. Antisera against link protein synthetic oligopeptides were obtained from rabbits immunized four times with 0.2 mg peptide conjugated to keyhole limpet hemocyanin together with Freund's adjuvant. Titration of antisera was performed by an ELISA, with peptides used for immunization as antigen. When the antibody titer reached a plateau, blood was collected and the serum was separated. Polyclonal antibodies against NH2-terminal synthetic oligopeptides of LP (anti-LPpep-N) and against COOH-terminal peptides of LP (anti-LPpep-C) were prepared using the elutant from protein GSepharose (Hitrap; Pharmacia). Polyclonal antibodies raised against link protein synthtic peptides reacted under nonreducing conditions in immunoblot analyses and in ELISA with purified bovine link protein. These domain-specific antibodies reacted with bovine cartilage tissue (not shown) but reacted only weakly with mouse granulosa cell-derived link protein in cryostat sections and in formalin-fixed, paraffin-embedded sections.
Enzyme-linked Immunosorbent Assay
Anti-LPpep-N (2 µg/ml) was applied in 100-µl aliquots to wells of a polystyrene microtiter plate (Nunc; Roskild, Denmark) and incubated overnight at 4C. After three washes with Tris-buffered saline (TBS) supplemented with 0.05% Tween-20 (TBS-T), residual protein binding sites were saturated by incubating each well with 200 µl of TBS containing 2% BSA. The blocking solution was aspirated, the wells were washed twice with TBS-T, and then 100 µl of the sample (10-fold dilution) was placed in each well overnight at 4C. After seven washes with TBS-T, the plates were incubated with biotinylated anti-LPpep-C antibody (1 µg/ml; 100 µl/well). After seven washes with TBS-T, 100 µl of avidinperoxidase (Dako, Glostrup, Denmark; 1:4000 in TBS-T/0.5 mol/liter NaCl) was added for 1 hr at 23C. After seven washes, binding of avidinperoxidase was detected at 23C by using 50 µl of solution containing tetramethylbenzidine (1 mg/ml) and 0.0003% H2O2 as the substrate for peroxidase. The reaction was stopped after 10 min by addition of 50 µl of 1 mol/liter H2SO4. The yellow absorbance was measured at 450 nm on a microtiter plate reader.
NH2-terminal Sequence of Immunoreactive Link Protein Bands
A sample of 50 pmol of the 49-kD and 40-kD bands (Figure 1) was sequenced in an Applied Biosystems 475A gas-phase automated sequencer and the amino acid sequence was analyzed.
Animals and Tissue Preparations
Female mice were purchased from SLC (Shizuoka, Japan) and were housed in a temperature-controlled room with a 12-hr light, 12-hr dark schedule and were fed chow and water ad libitum. Twenty-one-day-old immature female mice were treated with IP injection of 5 IU pregnant mare serum's gonadotropin (PMSG; Sigma) in 0.1 ml PBS, pH 7.4. Mice were treated with IP injection of 5 IU human chorionic gonadotropin (hCG; Sigma) 44 hr later, and ovaries were removed 1, 2, 4, 8, 12, or 24 hr later and used for further immunohistochemical experiments and Western blotting.
For immunoblot analysis, dissected ovaries were rinsed in PBS and then homogenized in 8 M urea, 50 mM sodium acetate, pH 5.8, and 50 U/ml Streptomyces hyaluronidase (Seikagaku Kogyo; Tokyo, Japan) supplemented with 0.2 mM [4-(2-aminoethyl)-bensenesulfonylfluoride, HCl] (Calbiochem, LaJolla, CA), 1 mg/ml approtinin, 10 µg/ml leupeptin, 10 µg/ml pepstatin (Boehringer; Mannheim, Germany), 1 mM benzamidine, and 1 mM phenylmethylsulfonyl fluoride (Sigma) as protease inhibitors, using a Polytron homogenizer. The amount of protein in the soluble fraction was quantified in a Bradford assay (Bio-Rad; Hercules, CA) using bovine serum albumin as a standard (
Immunohistochemistry
Localization of link protein was examined immunohistochemically using the polyclonal antibodies for bovine link protein. Specimens were placed in OCT compound (Sankyo; Tokyo, Japan), frozen with liquid nitrogen, and stored at -70C until sectioning and then were cut into sections about 10 µm thick. The cryostat sections were air-dried overnight and fixed with 2.5 % (w/v) paraformaldehyde in 0.1 M PBS. Some sections were fixed after digestion with Streptomyces hyaluronidase. The sections were treated with 50 U/ml Streptomyces hyaluronidase in 50 mM sodium acetate containing 0.15 M NaCl, pH 5.0, for 1r h at 23C.
In a parallel experiment, mouse ovaries were fixed in 4% (w/v) paraformaldehyde in 0.1 M PBS. The tissues were fixed at 23C overnight with shaking and washed in 0.1 M phosphate buffer containing 0.25 M sucrose and 0.2 M glycine, pH 7.4, several times over a period of 16 hr. After passing through a series of graded alcohol and xylene solutions, the tissues were embedded in paraffin by standard procedures. Five-µm sections were taken on glass slides for immunostaining. Hematoxylin-stained sections were used for identification of follicles and cumulus cells. The cryostat sections and the deparaffinized and rehydrated tissue sections were immersed in 0.3% H2O2 in methanol to block endogenous peroxidase and were preincubated with 5% (w/v) BSA in PBS for 1 hr at 23C to block nonspecific binding. The sections were reacted with the polyclonal antibody against link protein (5 µg/ml) diluted with 2% BSA in PBS for 16 hr at 4C in a humidified atmosphere. After washing in PBS three times for 15 min, the specimens were incubated with biotin-conjugated secondary antibodies (1:200; Dako) diluted with 2% BSA in PBS for 30 min at 23C. The specimens were then washed three times with PBS and incubated with avidinperoxidase (Dako), diluted 1:100 with 2% BSA in PBS for 30 min at 23C. They were washed three times with PBS. Peroxidase activity was seen after incubation in 100 mM TBS containing 0.03% H2O2 and 0.05% diaminobenzidine tetrahydrochloride. All sections were washed repeatedly with PBS and counterstained with hematoxylin. As a control, some of the sections were reacted with rabbit nonimmune IgG in place of the specific antibodies, and some were incubated with the primary antibody in the presence of an excess amount of purified link protein (100-fold at a molar ratio).
Immunostaining was assessed semiquantitatively as the intensity and percentage of positively stained cells (see Table 1 legend).
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Immunodetection of Link Protein in Extracts from Whole Ovaries
For each tissue, 20 µg of total protein was mixed with the SDS sample buffer (5% SDS, 10% glycerol), boiled for 5 min, separated by SDS-PAGE with a 10% gel according to Laemmli's method (1970), and electrophoretically transferred onto polyvinylidine difluoride (PVDF) membranes (Immobilon; Millipore, Bedford, MA). The membranes were blocked for 1 hr in TBS with 2% BSA and incubated for 2 hr with 1:500 polyclonal antibodies raised against link protein and then for 1 hr with biotinylated goat anti-rabbit IgG as the second antibody (1:500, 1 hr, 23C; Dako), followed by avidinperoxidase (1:500, 1 hr, 23C; Dako). Bands were visualized with the ECL detection system (Amersham Japan; Tokyo, Japan). Briefly, the PVDF membranes were incubated for precisely 1 min in a mixture of 5 ml of each of the ECL detection reagents. The membranes were then placed between two transparencies and exposed to Kodak film. In all experiments, some strips were incubated with nonimmune rabbit IgG as a negative control.
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Results |
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Purification of Link Protein
For purification of link protein, the HA binding protein was further fractionated by gel filtration. Aliquots of each fraction were tested for immunoreactivity by a specific ELISA for link protein and a crude link protein peak was obtained. Fractions containing immunoreactive link protein were dialyzed and a link protein peak was obtained. The link protein was further purified by anti-LPpep-N-coupled Sepharose 4B. Eluted materials were analyzed by SDS-PAGE followed by Western blotting using anti-link protein antibodies (Figure 1). The purified link protein revealed double bands of 49 kD and 40 kD under nonreducing conditions on Western blotting.
The link protein purified from HA binding protein does not contain an HA binding region (HA-BR) within the aggrecan molecule, which was confirmed by a specific ELISA (Aggrecan ELISA kit; BioSource Europe, Nivelles, Belgium) and Western blot analyses with specific monoclonal antibodies raised against HA-BR within aggrecan (Cosmo Bio; Tokyo, Japan) (unpublished data; and
The NH2-terminal amino acid sequence of the purified proteins was determined by automated gas-phase sequencing. These proteins exhibited a single NH2-terminal sequence. The first five amino acids of the 49-kD protein were analyzed. This polypeptide [16DHHSD20 (
Specificity of the Antibody
The polyclonal antibody to link protein was raised against link protein purified from the trypsin-generated HA binding protein of bovine nasal cartilage proteoglycan aggregate. Specificity of the antibody was studied by Western blot assay. Immature mice (21 days old) were treated with IP injection of hCG 44 hr after PMSG injection, and ovaries were removed 12 hr later. Total protein extracts prepared from 21-day-old immature mouse ovary and gonadotropin-treated immature ovary were tested for the presence of immunoreactive link protein by gel electrophoresis and immunoblotting. On the Western blotting analysis, as shown in Figure 1, anti-link protein antibody reacted not only with the purified link protein (Figure 1, Lane 1) but also with the homogenate of gonadotropin-treated immature mouse ovary (Figure 1, Lane 5). A reacting protein species of about 42 kD was seen in the gonadotropin-treated ovarian extract (Figure 1, Lane 5). However, the extract prepared from immature mouse ovary did not contain the immunoreactive link protein (Figure 1, Lane 4). The anti-link protein antibody reacts with purified link protein, forming double protein bands with molecular weights of 49 and 40 kD (Figure 1, Lane 1). It was confirmed that the 40-kD protein is a degradation product of the 49-kD link protein. Treatment of the link protein with Streptomyces hyaluronidase (Figure 1, Lane 2) or chondroitinase ABC (Figure 1, Lane 3) did not lead to an increase in the mobility of this species. This result indicated that the present anti-link protein antibody specifically detects link protein contained in the gonadotropin-treated mouse ovary and that link protein itself is insensitive to both hyaluronidase and chondroitinase. Control slides incubated with nonimmune rabbit serum as primary antibody revealed no staining (not shown). In addition, incubation with the primary antibody in the presence of an excess amount of purified link protein revealed no bands (not shown).
Immunohistochemical Analysis of Link Protein Expression in Mouse Ovary
We tested the antibodies for their suitability for immunohistochemical studies. First, we compared formalin-fixed, paraffin-embedded tissue sections with cryostat sections obtained from the same ovary. The patterns of immunostaining of anti-link protein antibody on paraffin sections were superimposable with those of cryostat sections; it is improbable that staining was more pronounced in cryostat sections (not shown). The intensity of staining was completely suppressed by preincubation of the cryostat sections with hyaluronidase before fixation.
Localization of link protein was immunohistochemically analyzed in the ovary from untreated (Figure 2) and gonadotropin-treated immature mice (Figure 3 Figure 4 Figure 5 Figure 6) in the fixed cryostat sections. At 21 days of life, a few antral follicles were seen in addition to the primordial and primary follicles, and the interstitial glands were developed. Intense and granular link protein staining was found in oocytes in ovaries taken from 21-day-old mice before PMSG treatment (Figure 2A). All of the oocytes, when present on the sections, were always stained. There was detectable reaction product also in theca cells. In the antral follicle stage, immunostaining for link protein became apparent in only a fraction of the granulosa cells, whereas the granulosa cells in the primary or preantral follicles were not immunostained with anti-link protein antibody. In the control experiments, replacement of the primary antibody with nonimmune IgG showed no positive immunostaining of oocyte and theca cells for link protein (Figure 2B). To ascertain whether link protein exists in association with HA, cryostat sections were left unfixed, immediately treated with Streptomyces hyaluronidase, and finally fixed. This led to almost complete loss of link protein reactivity (Figure 2C). Sections treated in an identical manner, but with the enzyme omitted, retained normal levels of reactivity for the link protein (Figure 2A).
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It is well established that treatment of immature mice with gonadotropins gives rise to a synchronized process of follicle development, ovulation, and corpus luteum formation in the ovary. The follicles of immature mice ovaries are induced to develop into large preovulatory (Graafian) follicles by 44 hr after administration of PMSG, and subsequent treatment with hCG brings about a synchronized ovulation of many follicles that peaks around 12 hr after administration of hCG. Our finding was consistent with the previous reports (
Twenty-four hr after hCG treatment, the ovary was filled with corpora lutea in their developing stages. Almost all the luteal cells were immunostained with anti-link protein antibody (Figure 5A). The ovulated COC was found in the distended proximal portion of the oviduct (Figure 6C). Positive link protein staining was seen in the oviductal epithelial cells (Figure 6A and Figure 6B) as well as in the oocytes and the cytoplasm of the cumulus cells (Figure 6C).
Table 1 summarizes the results of immunohistochemistry for link protein in mouse ovary. Throughout the course of follicle maturation, ovulation, and luteinization induced by the gonadotropin treatment, link protein immunoreactivity was localized exclusively in the theca cells. Oocytes were also positive for link protein antigen, irrespective of follicle developmental stage. The cumulus cells (both in the cytoplasm and in the extracellular matrix) and mural granulosa cells (in the cytoplasm) in the preovulatory follicles were essentially positive for link protein antigen, and signal intensity was variable among the cells. The intensity of immunostaining for link protein observed in the granulosa cells increased as the follicle became larger and matured. Almost all luteal cells in the corpus luteum displayed immunostaining for link protein.
The immunolocalization of link protein in all oocytes in ovaries and in granulosa cells of antral follicles from immature animals not treated with PMSG is inconsistent with the absence of link protein in ovaries of untreated animals shown by Western blotting (Figure 1, Lane 4). It is unlikely that oocyte staining is nonspecific because it was not present in controls. These contradictory data may be reconciled by the finding that weak but positive staining (a 42-kD band) was obtained by incubation of the antibody with 100 µg total protein extract of untreated ovary (not shown).
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Discussion |
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I protein binds HA, it stabilizes the extracellular matrix of the mouse COC. Localization and function of HA and proteins of the IaI family were studied in mouse (
With the immunohistochemical approach, it is possible to determine the specific tissue compartments that express link protein antigen during follicle development (Figure 2 Figure 3 Figure 4 Figure 5 Figure 6). The results of this study showed that (a) immunoreactivity for link protein appeared on the theca cells and oocytes not only of primordial and primary follicles but also of secondary and large preovulatory follicles, irrespective of the mouse age and hormonal condition, and that (b) there was a periovulatory increase in granulosa cell-derived link protein staining of gonadotropin-treated immature animals. Highest levels of link protein immunoreactivity were observed in the cumulus cells and mural granulosa cells of the large preovulatory follicles. (c) Link protein expression persisted in luteinized granulosa cells after ovulation and in corpus luteum, and (d) addition of hyaluronidase to the specimens led to disappearance of the link protein. This enzyme is HA-specific because it does not attack other glycosaminoglycans (I in the COC matrix and hence are required for successful COC expansion, because the I
I heavy chains have been shown to interact with link protein independently of HA (
The localization of link protein was similar to those of HA (I (
It appears unlikely that mural granulosa cells and cumulus cells differ in their ability to produce link protein. The cumulus cells synthesize and deposit an intercellular matrix enriched in HA, which leads to expansion of the COC (
Link protein possibly produced by cumulus cells could also help to trap HAII complexes in the extracellular matrix on the COC, and could therefore directly affect stabilization of HAI
I complexes. We believe, therefore, that in these locations the extracellular matrix of cumulus cells consists, at least in part, of the HAlink proteinI
I complexes and that formation of ternary complexes in this compartment is related to and strengthens COC expansion. It has been shown that HA and I
I exhibit a cytoplasmic and extracellular distribution in the maturing mouse ovary (
I (
The high level of link protein apparent in the mural granulosa cells 24 hr after hCG may be temporally related to the rapid transformation of the follicle into a highly vascularized corpus luteum. The present immunocytochemical study clearly shows the existence of two regions in the corpus luteum: the internal region, probably corresponding to granulosa-derived cells, which contains link protein, and the external region, probably of thecal origin, which also contains this protein. This demonstrates that the link protein-positive mural granulosa cells and theca cells remain in the follicle and contribute to the process of corpus luteum formation.
Link protein production in oviductal epithelium has not been observed previously. The marked epithelial link protein staining in the ampulla region of the oviduct at the time when COC were present implies that oviductal link protein synthesis could be important in supplying this protein to the COC. The presence of link protein in oocytes, cumulus cells, and surrounding oviductal epithelium may indicate a role for link protein in the transfer of eggs in the oviduct.
In conclusion, this is the first study to demonstrate a marked change in the expression of link protein in the granulosa and cumulus cells of the preovulatory follicle. The role of the link protein appears to be in crosslinking of II by permitting a more stable interaction with HA.
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Acknowledgments |
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We thank Prof Dr M. Terada (Animal Center, Hamamatsu University School of Medicine), Drs E. Morishita and K. Kato (BioResearch Institute, Mochida Pharmaceutical Co., Tokyo), Drs M. Ikeda and S. Miyauchi (Seikagaku Kogyo, Tokyo), and Drs Y. Tanaka and T. Kondo (Chugai Pharmaceutical Co., Tokyo) for their continous and generous support of our work.
Received for publication January 6, 1999; accepted May 19, 1999.
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