Journal of Histochemistry and Cytochemistry, Vol. 47, 1433-1442, November 1999, Copyright © 1999, The Histochemical Society, Inc.


ARTICLE

Expression of Link Protein During Mouse Follicular Development

Guang W. Suna, Hiroshi Kobayashia, and Toshihiko Teraoa
a Department of Obstetrics and Gynecology, Hamamatsu University School of Medicine, Hamamatsu, Shizuoka, Japan

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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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 47:1433–1442, 1999)

Key Words: cumulus oocyte complex, expansion, hyaluronic acid, inter-{alpha}-trypsin inhibitor, link protein


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The development of ovarian follicles is considered to be regulated by various factors such as gonadotropins (Eppig 1979 ), cytokines, growth factors, and locally produced hormones (Salustri et al. 1990a ). Gonadotropin triggers cumulus–oocyte complex (COC) expansion and induces ovulation (Dekel et al. 1979 ; Eppig 1979 ; Salustri et al. 1992 ). Several lines of evidence suggest that hyaluronic acid (HA) plays an important role in the gonadotropin induction of COC expansion and that oocytes produce a factor which stimulates HA synthesis by cumulus cells (Salustri et al. 1990b ). A natural ligand for HA has been discovered and identified as a 240-kD inter-{alpha}-trypsin inhibitor (I{alpha}I) (Chen et al. 1992 , Chen et al. 1994 ; Huang et al. 1993 ; Castillo and Templeton 1993 ; Jessen et al. 1994 ; Zhao et al. 1995 ). Several articles have been devoted to study of the molecular mechanisms responsible for the initiation, progression, and maintenance of HA-rich matrix on the cumulus cells, demonstrating that COC expansion is induced through HA production and accumulation of proteins of the I{alpha}I family in the gonadotropin-stimulated cumulus cells (Chen et al. 1992 , Chen et al. 1994 ). These findings are very similar to the biochemical evidence indicating that the complexes involving HA and proteoglycans are an important component of the extracellular matrix of cartilage (Poole et al. 1980 ).

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 (Camaioni et al. 1996 ). The ~46-kD protein has approximately the same molecular size as the link protein that interacts with HA and HA-binding proteoglycans to form stable ternary complexes in a variety of extracellular matrices. Link protein is reported to be composed of glycoproteins of 44.5 and 48.5 kD (Bonnet et al. 1978 ; Tang et al. 1979 ), which stabilize the binding between proteoglycan subunits and HA. Link protein binds simultaneously to the HA binding region of the proteoglycan molecule and to HA itself (Hardingham and Muir 1974 ). In addition, link protein binds to I{alpha}I (Hirashima et al. 1997 ). Because link protein is mainly found in the extracellular matrix, it is believed to be involved in the organization of an HA-containing matrix. We and others (Camaioni et al. 1996 ) therefore speculate that link protein may further stabilize the binding between HA and I{alpha}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 Camaioni et al. 1996 corresponds to the link protein. Available data on the local production of the ~46-kD protein in the ovary are based on examinations focused on a limited experiment by Camaioni et al. 1996 . Furthermore, there is no information on the expression and localization of the link protein molecule in maturing ovaries. For a better understanding of the physiology of COC expansion, analysis of the expression of link protein in individual cells would be required. We have set up an in vivo model to study the gonadotropin induction of link protein levels in pregnant mare's serum gonadotropin (PMSG)-primed human chorionic gonadotropin (hCG)-stimulated immature mice and have correlated ovarian morphological development with changes in cell localization of link protein antigen. For this purpose, polyclonal antibodies were raised against link protein purified from bovine cartilage.


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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 (Baker and Caterson 1979 ; Neame and Barry 1994 ). A purified preparation of HA binding protein was a generous gift from Drs. S. Miyauchi and M. Ikeda (Seikagaku Kogyo; Tokyo, Japan) and from Drs. Y. Tanaka and T. Kondo (Chugai Pharmaceutical; Tokyo, Japan). In brief, bovine nasal cartilage link protein was prepared by dissociative extraction of the cartilage in 4 M guanidine-HCl plus protease inhibitors [10 mM benzamidine, 1 mg/ml leupeptin, 1 mM phenylmethyl sulfonylfluoride, 0.1 mg/ml ovalbumin, and 0.1 unit/ml aprotinin; all reagents from Sigma (St Louis, MO)], reassociation of the proteoglycan–HA aggregate, and isopycnic cesium chloride gradient centrifugation. The bottom fraction containing the aggregate (A1) was dissociated in 4 M guanidine-HCl and subjected to a further centrifugation step, and the top fraction, containing link protein, (A1D4) was isolated as described by Baker and Caterson 1979 .

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 Tang et al. 1979 . The crude LP was fractionated by HPLC gel filtration using an SW3000 column (Kanto Kagaku; Tokyo, Japan). Aliquots of each fraction were tested for their immunoreactivity by a specific enzyme-linked immunosorbent assay (ELISA; see below) and a link protein peak was obtained. The link protein was further purified by anti-LPpep-N= coupled Sepharose 4B. Briefly, purified anti-LPpep-N antibody (10 mg; see below) was coupled to CNBr-activated Sepharose 4B (3 g dry weight = 10 ml bed volume) (Pharmacia; Uppsala, Sweden) according to the manufacturer's recommendations. The crude link protein fraction was dialyzed and mixed with anti-LPpep-N antibody–Sepharose 4B previously equilibrated in 20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.2% Triton X-100 containing protease inhibitors, using end-to-end rocking for 16 hr at 4C. The affinity gel was then washed 10 times with 20 mM Tris-HCl, pH 7.4, containing 150 mM NaCl and twice with H2O. The column was eluted with 50 mM glycine-HCl, pH 3.0. Eluted materials were dialyzed and a purified link protein peak (49-kD and 40-kD double bands by Western blotting; see Figure 1) was obtained.



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Figure 1. Immunodetection of link protein on Western blots of purified link protein, digested link protein, or total protein extracts Purified link protein (2 µg) was treated with hyaluronidase (final concentration 50 U/ml; Lane 2) or chondroitinase ABC (0.1 µg; Lane 3) for 1 hr at 23C. Link protein was left untreated (Lane 1, 2 µg/lane). The samples as well as total protein extracts prepared from 21-day-old immature ovary (Lane 4, 20 µg/lane) and gonadotropin-treated ovary (Lane 5, 20 µg/lane) were run in a 10% gel and transferred to PVDF sheets. The blot was labeled with the anti-link protein antibody and detection was achieved using the ECL Western blotting detection reagents. Anti-link protein polyclonal antibody recognizes double protein bands with molecular weights of 49 and 40 kD. Incubation of the extracts with anti-link protein antibody demonstrated immunoreactive protein (42 kD) in gonadotropin-treated ovary but not in immature ovary. MW, molecular weight markers. In addition, 100 µg of tissue lysates showed staining of a 42-kD single band even in an immature ovary.

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 aggrecan–HA binding region (unpublished data; and Kobayashi et al. 1998 ), suggesting that this antiserum was specific for bovine cartilage link protein.

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 G–Sepharose (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 avidin–peroxidase (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 avidin–peroxidase 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 (Bradford 1976 ).

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 avidin–peroxidase (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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Table 1. Immunohistochemical localization of link protein in various cell types of mouse ovarya

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 avidin–peroxidase (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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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 Kobayashi et al. 1998 ).

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 (Neame and Barry 1994 )] has been sequenced and was identical to sequences obtained from bovine link protein. In addition, to explore the possibility that the polypeptide band at Mr 40 kD is also related to link protein, the primary structure of the 40-kD polypeptide was determined. This polypeptide (39RLLVEAEQAKV49) was identical to sequence 39–49 of the bovine link protein.

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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Figure 2. Immunohistochemical localization of link protein in the 21-day-old immature ovary. Sections fixed with paraformaldehyde, 10-µm-thick, hematoxylin-counterstained sections. (A) Before PMSG treatment. Sections of mouse ovary were labeled with the anti-link protein antibody. Intense immunostaining for link protein was present in the oocyte, irrespective of the stage of follicular growth. The theca cells also immunostained weakly. (B) Replacement of primary antibody with nonimmune IgG showed no positive immunostaining of the theca cells and oocytes for link protein. (C) Serial sections were digested with 50 U/ml Streptomyces hyaluronidase for 1 hr at 23C before fixation. Labeling for link protein was abolished by hyaluronidase. f, follicular fluid; g, granulosa cells; o, oocyte; t, theca cells. Bars = 20 µm.

Figure 3. Immunohistochemical localization of link protein in primary, preantral, and antral follicles. Sections fixed with paraformaldehyde, 10-µm-thick, hematoxylin-counterstained sections; 2 hr after hCG treatment. The sections were labeled with the anti-link protein antibody. The immunostaining for link protein observed in the granulosa cells increased as the follicle developed. Only a fraction of the granulosa cells contained link protein immunnoreactivity. Oocyte and theca cells show link protein staining. (A) Primary follicle; (B) preantral follicle; (C) antral follicle. cl, corpus luteum; f, follicular fluid; g, granulosa cells; o, oocyte; t, theca cells. Bars = 20 µm.



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Figure 4. Immunohistochemical localization of link protein in preovulatory follicles. Sections fixed with paraformaldehyde, 10-µm-thick, hematoxylin-counterstained sections; 12 hr after hCG treatment. Sections of mouse ovary were labeled with the anti-link protein antibody. (A,C) Ovarian sections contain marked link protein immunoreaction in the cumulus cells (in the cytoplasm and in the extracellular matrix) and mural granulosa cells (in the cytoplasm). Oocyte and theca cells also show link protein staining. (B,D) Replacement of primary antibody with nonimmune IgG showed a lack of positive immunostaining. f, follicular fluid; mg, mural granulosa cells; t, theca cells; arrow, COC; triangle, oocyte. Bars = 20 µm.

Figure 5. Immunohistochemical localization of link protein in corpus luteum. Sections fixed with paraformaldehyde, 10-µm-thick, hematoxylin-counterstained sections; 24 hr after hCG treatment. Sections of mouse ovary were labeled with the anti-link protein antibody. Ovarian sections contain link protein immunoreaction in the cytoplasm of the corpus luteum (A). Replacement of primary antibody with nonimmune IgG showed no positive immunostaining (B). cl, corpus luteum. Bars = 20 µm.



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Figure 6. Immunohistochemical localization of link protein in oviduct. Sections fixed with paraformaldehyde, 10-µm-thick, hematoxylin-counterstained sections. Sections of mouse ovary were labeled with the anti-link protein antibody. Oviduct sections contain link protein immunoreaction in the cytoplasm of epithelial cells 8 hr after hCG treatment (A). Positive reaction product for link protein was observed in the distended proximal portion of the oviduct 24 hr after hCG treatment (B), in which the ovulated COCs were found (C). Ovulated COCs found in the oviduct show positive staining for link protein. e, epithelial cells; o, oocyte; lumen of the oviduct; arrow, COC. Bars = 20 µm.

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 (Fischer and Fischer 1975 ). PMSG treatment stimulated follicle development. By 44 hr after PMSG administration, maturation of follicles had further progressed and some large follicles with abundant follicular fluid were seen. Two hr after hCG injection, positive link protein staining was still observed in oocytes and theca cells (Figure 3). It is unlikely that the staining intensity of the oocyte increased as the follicle developed. In contrast, the granulosa cells of primary follicles were completely devoid of immunoreactivity (Figure 3A). The immunostaining for link protein observed in the granulosa cells increased as the follicle developed (Figure 3B and Figure 3C). However, only a fraction of the granulosa cells contained link protein immunoreactivity. Immunoreactivity for link protein was recognized in the majority of the granulosa cells of large follicles (not shown). At 8 hr after hCG, intense link protein immunostaining appeared in the granulosa layer (in the cytoplasm and into the intercellular matrix) (not shown). Close to ovulation time (12 hr after hCG treatment following 44 hr PMSG treatment), the highest levels of link protein reaction pro–duct were observed in mural granulosa cells of pre–ovulatory follicles and in cumulus cells (Figure 4A and Figure C). Close to ovulation, immunoreactive link protein is of a very wide range, although the staining intensity in Figure 4A (close to ovulation) seems to be no greater than that in Figure 3A. The link protein staining reaction was diffusely concentrated in the cytoplasm of the mural granulosa cells (Figure 4C). It was obvious that the reaction product was always found in the cytoplasm and was usually located in the intercellular matrix of the cumulus cells (Figure 4C). Most of the cells in the theca interna cells surrounding preovulatory follicles contained cytoplasmic link protein staining. Positive link protein immunoreactivity was also observed in the theca cells around most small to middle-sized nonovulatory follicles. Therefore, theca cells displayed a similar staining all around the immature and mature follicles. Replacement of primary antibody with nonimmune IgG showed no positive immunostaining of oocyte, theca cells, and granulosa cells for link protein (Figure 4B and Figure 4D). Treatment of the sections with hyaluronidase abolished the immunostaining of the follicular compartment for link protein (not shown). Therefore, the cytoplasmic and extracellular labeling seen in the granulosa cells with the anti-link protein antibody was sensitive to hyaluronidase.

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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Literature Cited

Chen et al. 1992 were the first to assign a role to the interaction showing that, when an I{alpha}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 (Chen et al. 1992 ; Castillo and Templeton 1993 ) and bovine (Eppig 1979 ; Camaioni et al. 1993 ) ovaries, and the distribution patterns of these antigens was very similar. Recently, Camaioni et al. 1996 reported that the ~46-kD protein (which appears to be link protein because of it molecular weight) forms complexes that are necessary for retaining HA in the COC matrix. Therefore, it was of interest to determine how link protein is distributed in mouse ovary. For this purpose, we raised polyclonal antibodies against bovine link protein and its synthtic oligopeptides and studied the localization of this protein in mouse ovary by immunocytochemical techniques. We have shown that the polyclonal antibodies against bovine link protein produced by us recognized the 42-kD protein in the gonadotropin-treated mouse ovary (Figure 1), the size of which was similar to those of the cartilage link proteins (49-kD and 40-kD). Therefore, the polyclonal antibody used in this study recognizes an antigenic determinant present in link protein obtained from bovine and mouse species. However, no experiments have been described that demonstrate that the antibody specifically interacts only with mouse link protein, although it reacts with a 42-kD band in the stimulated mouse ovary.

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 (Ohya and Kaneko 1970 ). Link protein itself is not sensitive to hyaluronidase or chondroitinase (Figure 1). This suggests that the release was dependent on the degradation of HA rather than being brought about by proteolytic activity. The outcome of this experiment makes it very probable that link protein is bound to HA. These results indicate for the first time that granulosa cell-derived link protein is increased in association with follicle development and support the hypothesis that link protein is synthesized by the cumulus cells to form stable complexes that are necessary for retaining both HA and I{alpha}I in the COC matrix and hence are required for successful COC expansion, because the I{alpha}I heavy chains have been shown to interact with link protein independently of HA (Hirashima et al. 1997 ).

The localization of link protein was similar to those of HA (Salustri et al. 1990a , Salustri et al. 1990b , Salustri et al. 1992 ) and I{alpha}I (Chen et al. 1992 ) previously reported. However, the localization was somewhat different from that of HA, because oocytes failed to express HA. The presence of detectable link protein even in the theca cells and oocytes of the immature follicle suggests that it is not a target of direct gonadotropin stimulation. The results obtained do not support link protein participation in oocyte maturation. It is also possible that theca cell-derived link protein may have a function other than the stabilization of proteoglycan aggregates, e.g., the role of these cells may be related to sustaining transportation of link protein as a source.

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 (Dekel et al. 1979 ). However, the mural granulosa cells do not elaborate a similar intercellular matrix because of negligible production of HA (Salustri et al. 1990a ). Consequently, the mural granulosa cells remain adherent to the outer basement membrane. This may be due to their different proximities to the oocyte rather than to their different ability to synthesize link protein or HA (Salustri et al. 1990a ).

Link protein possibly produced by cumulus cells could also help to trap HA–I{alpha}I complexes in the extracellular matrix on the COC, and could therefore directly affect stabilization of HA–I{alpha}I complexes. We believe, therefore, that in these locations the extracellular matrix of cumulus cells consists, at least in part, of the HA–link protein–I{alpha}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{alpha}I exhibit a cytoplasmic and extracellular distribution in the maturing mouse ovary (Chen et al. 1992 ; Salustri et al. 1992 ), that link protein is considered to be synthesized by mural granulosa and cumulus cells, and that link protein is bound to both HA and I{alpha}I (Kobayashi et al. 1998 ). These may be the reasons why so much of the detected link protein is in the cytoplasm of mural granulosa cells, although link protein is essentially an extracellular matrix protein in cumulus cells. Alternatively, it is possible that the ternary complexes are endocytosed by the cells.

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 I{alpha}I by permitting a more stable interaction with HA.


  Acknowledgments

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.


  Literature Cited
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Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

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