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Biology of Reproduction 67, 1165-1171 (2002)
© 2002 Society for the Study of Reproduction, Inc.

Identification of Hyaluronic Acid-Binding Proteins and Their Expressions in Porcine Cumulus-Oocyte Complexes During In Vitro Maturation1

Masaki Yokoo2,a, Yasunori Miyahayashia, Takako Naganumab, Naoko Kimuraa, Hiroshi Sasadaa, and Eimei Satoa

a Laboratory of Animal Reproduction, Graduate School of Agricultural Science, Tohoku University, Sendai 981-8555, Japan b Laboratory of Functional Biomolecules, Department of Biomolecular Science, Graduate School of Life Science, Tohoku University, Sendai 981-8555, Japan


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Hyaluronic acid-binding proteins (HABPs) are necessary for expansion of the cumulus-oocyte complex (COC) during oocyte maturation. In this study, to obtain the detailed information of HABPs during cumulus expansion, we examined the expression of HABPs in porcine COCs during in vitro maturation (IVM). After maturation culture, proteins were extracted from porcine COCs and separated by SDS-PAGE and then transferred to polyvinylidene fluoride membranes. After transfer, the membranes were subjected to ligand blotting with biotinylated hyaluronic acid (bHA) or fluorescein isothiocyanate-labeled hyaluronic acid (FITC-HA). Furthermore, the extracted proteins were subjected to immunoprecipitation, Western blotting, and immunofluorescence analysis to dissect the HABPs. Ligand blotting with FITC-HA could detect HABPs. Using this ligand-blotting method, 13 and 14 bands of HABPs were detected in porcine COCs after 0 and 48 h in culture, respectively. Of these, the level of expression of 85-kDa HABP increased with cumulus expansion during IVM and was newly detected after culture. Immunoprecipitation, Western blotting, and immunofluorescent analysis confirmed that the 85-kDa HABP corresponded to CD44 and that it existed on/in the membrane of cumulus cells. The present results indicated that HABP expressed in porcine COCs during IVM, particularly CD44, may form a network of the matrices in the extracellular space of the oocyte with cumulus expansion during IVM.

cumulus cells, granulosa cells, oocyte development, ovary, ovum


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
During the preovulatory period, cumulus expansion occurs dramatically in cumulus-oocyte complexes (COCs), where large amounts of glycosaminoglycans (GAGs) are synthesized and secreted. In turn, these GAGs interact with specific matrix components, thereby forming a network of highly hydrated and viscoelastic matrices in the extracellular space. Cumulus expansion facilitates detachment of the COC from the follicle wall, its extrusion at ovulation [1–3], and its capture by oviductal fimbria [4] and promotes the acrosome reaction in spermatozoa [5, 6]. Furthermore, cumulus expansion may influence a variety of fundamental developmental changes, which occur during fertilization between an oocyte and a spermatozoon, followed by subsequent development of a zygote [1, 7, 8]. Similarly to COC expansion within the follicle in vivo, COCs are expanded during in vitro maturation (IVM) by the influence of gonadotropins.

The main component of cumulus expansion is hyaluronic acid (HA) [8–11], and the amount of HA synthesized is closely correlated with the degree of cumulus expansion [12, 13]. Consequently, HA is thought to be involved in these functions. HA is a linear GAG that is a high molecular weight polymer with repeating disaccharide units of sodium glucuronate and N-acetyl glucosamine linked by ß 1–3 and ß 1–4 glycosidic bonds. Despite its structural simplicity, HA is a biologically important biopolymer that is widely distributed in the extracellular matrix of connective tissues in the body and plays important roles in diverse processes such as wound repair [14, 15], cell motility [16, 17], and cancer metastasis [18–21]. For this broad spectrum of biological activities, it is usually necessary to maintain the highly structured nature of HA. Unlike other GAGs, HA is neither sulfated nor linked to a core protein. Hence, HA needs hyaluronic acid-binding proteins (HABPs), an important subset of which have highly homologous sequences for HA binding, to form the extracellular HA-rich matrices [22]. This is likely true also in the formation of a three-dimensional cumulus cell-embedding structure in the expanded COCs during IVM. There is, however, less information regarding the dynamics of HABPs in porcine COCs during IVM to clarify the physiological roles of HA in cumulus expansion of COCs.

We recently reported the mRNA expressions of CD44, which is one of the HABPs, in porcine COCs during IVM [23]. In the present study, to obtain the more detailed information of HABPs in porcine COCs during IVM, we applied two ligand-blotting methods to detect HABPs when porcine COCs show cumulus expansion during IVM and demonstrated further identification of HABPs and their expressions during IVM.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Isolation and Culture of Porcine COCs

Porcine ovaries were obtained from prepubertal gilts at a slaughterhouse and carried to the laboratory within 30 min in a container kept at 37°C. Follicles 2–5 mm in diameter were aspirated with a 5-ml syringe with a 20-gauge needle and only the COCs that had uniform and compact cumulus cells were collected in modified TCM-199 (mTCM-199). Modified TCM-199 (Gibco BRG Life Technologies, Grand Island, NY) with Earle salt and L-glutamine contained 2.2 mg/ml sodium bicarbonate (Nacalai Tesque, Kyoto, Japan), 0.1 mg/ml sodium pyruvate (Sigma, St. Louis, MO), 10 mg/ml BSA (Sigma), 100 IU/ml penicillin (Meiji Seika, Tokyo, Japan), 100 µg/ml streptomycin (Meiji Seika), 10% (v/v) porcine follicular fluid, and 10 IU/ml eCG (Serotropin, Teikoku Zouki Pharmaceutical, Tokyo, Japan). The collected COCs were washed three times with mTCM199 and 50 COCs were cultured in 500 µl drops of the same medium covered with paraffin oil (Nacalai Tesque) for 48 h at 37°C under 5% CO2 in air.

Protein Extraction

At the end of culture, the COCs were transferred to microfuge tubes and were treated with hyaluronidase by vortexing for 15 min at 37°C. After treatment, the oocytes and the cumulus cells were washed three times with phosphate-buffered saline (PBS) and vortexed with 10 µl of cell lysis buffer (50 mM Tris, [pH 7.5], 1 mM phenylmethylsulfonyl fluoride, 0.1 M 6-amino-n-caproic acid, 5 mM benzamidine HCl, 1% [v/v] 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate). These agents were purchased from Sigma. After stirring at 4°C for 1 h, the tubes were centrifuged at 10 000 x g for 30 min and the supernatants were collected. The protein extracts were frozen and stored at -20°C until use.

Preparation of Biotinylated HA

HA was biotinylated essentially according to the method reported previously by Pouyani and Prestwich [24, 25] following the instructions for conjugation glycoproteins with ImmunoPure Biotin-LC-Hydrazide (Pierce Chemical, Rockford, IL). Briefly, 4 mg of HA was dissolved in 1 ml of PBS and dialyzed against 1 ml of MES buffer (0.1 M 2-N-morpholino ethanesulfonic acid [Sigma], pH 5.5) overnight at 4°C. Twenty microliters of 50 mM ImmunoPure Biotin-LC-Hydrazide, freshly dissolved in dimethyl sulfoxide (Sigma), was added to the HA solution. Freshly prepared EDC buffer (1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride [Pierce], 100 mg/ml in 0.1 M MES, pH 5.5) was then added to a final concentration of 13 µl/ml and stirred overnight at room temperature. The precipitate that formed during the reaction was removed by centrifugation. The resultant biotinylated HA (bHA) preparation was dialyzed against PBS at 4°C for 2 days before use.

Ligand Blotting

The extracted proteins were separated by 8% SDS-PAGE [26] under nonreducing conditions and electroblotted onto membranes in a semidry blotting apparatus according to the method of Hirano and Watanabe [27]. Electroblotting was performed for 90 min at a constant current of 0.8 mA/cm2. After electroblotting, the membrane was washed with PBS-T (0.05% [v/v] Tween 20, pH 7.4). The membrane was incubated with 10 µg/ml bHA for the bHA method or 10 µg/ml fluorescein isothiocyanate-conjugated HA (FITC-HA; CarboMer, Westborough, MA) for the FITC-HA method at 37°C for 1 h. The membranes probed with bHA were washed in PBS-T, then incubated with Streptavidin horseradish peroxidase (1:1000; Amersham, Arlington Heights, IL) for 1 h at room temperature. After washing three times with PBS-T, the peroxidase activity was visualized using the ECL Western blotting detection system (Amersham) according to the manufacturer's instructions. In the FITC-HA method, the membranes were washed three times with PBS-T and then the FITC signals were detected using an image analyzer (Molecular Imager FX Systems, Bio-Rad Laboratories, Hercules, CA).

Immunoprecipitation Analysis

Aliquots of 3 µg of anti-porcine CD44 monoclonal antibody (PORC24A; VMRD, Pullman, WA) were precoupled to 10 µl of protein G-sepharose (Amersham) by incubation for 1 h at 4°C. The COC extract (20 µl) was then added to the antibody-precoupled protein G beads and incubated for 2 h at 4°C. The extract was then centrifuged at 10 000 x g for 10 min, and the supernatant was collected and treated as the immunodepleted sample.

Cell Fractionation by Centrifugation

For cell fractionation, the COCs were washed three times in PBS after treatment with hyaluronidase and resuspended in PBS containing protease inhibitors. The COCs were chilled on ice for 5 min and then homogenized. After homogenization, the suspension was centrifuged at 600 x g for 10 min, and the supernatant was then recentrifuged at 8000 x g for 10 min. The resultant membrane pellet was lysed in cell lysis buffer at 4°C for 1 h. The lysed solution was centrifuged at 100 000 x g for 60 min, and the supernatant was collected and stored at -20°C until use as the membrane fraction. The pellet was used as the insoluble fraction.

Western Blotting

After electroblotting, the membranes were blocked with 2% skim milk in PBS-T overnight at 4°C and washed three times with PBS-T. The membranes were then incubated with PORC24A for 1 h at room temperature and then reacted with secondary antibody (horseradish peroxidase-labeled anti-mouse IgG; Sigma). After washing three times with PBS-T, the peroxidase activity was visualized using the ECL Western blotting detection system (Amersham) according to the manufacturer's instructions.

Immunofluorescence Analysis

At the end of cultivation, the COCs were fixed with 4% (w/v) paraformaldehyde-PBS (pH 7.4) at room temperature. After 30 min, they were rinsed three times with PBS and permeabilized with 0.5% (v/v) Triton X-100-PBS for 30 min at room temperature. The fixed COCs were incubated with PORC24A in 5% BSA in PBS for 2 h at 4°C, followed by rinsing with PBS and incubation with a fluorescein-conjugated goat anti-mouse IgG antibody (Sigma) at 37°C for 1 h. Propidium iodide staining was used as a control to stain nuclei. Finally, after several washes with BSA-PBS, the COCs were mounted on slide glasses, covered with a coverslip, and observed with a confocal microscope (MRC-1024, Bio-Rad). LaserSharp Processing software (Bio-Rad) was used to analyze the confocal images.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Detection of HABPs by Two Ligand-Blotting Methods

To identify HABPs in porcine COCs, we applied two ligand-blotting methods, the bHA method and the FITC-HA method (Fig. 1). Bovine nasal cartilage HABP (Seikagaku, Tokyo, Japan) was used as a positive control. Silver staining (Fig. 1, lane 1) showed the bovine nasal cartilage HABP as two major bands with mobility corresponding to known molecular weights of 40 and 45 kDa, respectively, which are two link proteins [28, 29]. Although these bands were detected by both ligand-blotting methods, the FITC-HA method was more sensitive than the bHA method (Fig. 1, lane 3 vs. lane 6). In the COC extraction, 9 and 14 bands of HABPs were detected by the bHA method and FITC-HA method, respectively (Fig. 1, lane 4 vs. lane 7). Furthermore, in the bHA method, two bands (70 and 120 kDa) were detected on the negative control membrane, which was incubated only with Streptavidin horseradish peroxidase (Fig. 1, lane 5).



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  		FIG. 1. Detection of HABPs by two ligand-blotting methods. Total proteins were visualized by silver staining (A), and HABPs were detected by ligand blotting using bHA (B) or FITC-HA (C) after transfer to membrane. The extracts of 50 porcine COCs were separated by 8% SDS-PAGE under nonreducing condition (lanes 2, 4, 5, 7). Bovine nasal cartilage HABP was used as a positive control (lanes 1, 3, 6). As a negative control, the membrane was incubated only with Streptavidin horseradish peroxidase (lane 5). The arrowheads indicate the HABP bands. The opened arrowheads indicate nonspecific bands in bHA method

Expressions of HABPs During IVM

After 0 and 48 h of culture, proteins were extracted from COCs and subjected to ligand blotting according to the FITC-HA method to detect HABPs. Thirteen bands of HABPs were detected in porcine COCs before culture (Fig. 2, lane 1), whereas 14 bands were detected after 48 h in culture (Fig. 2, lane 2). Of these, the 85-kDa HABP was newly expressed after culture for IVM.



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  		FIG. 2. Expressions of HABPs during IVM. The extracts were obtained from before (lane 1) and after cultivation (lane 2). The arrowheads indicate the HABP bands in porcine COCs. The closed arrowhead indicates the 85-kDa HABP

Identification of the 85-kDa HABP in Porcine COCs

CD44 is a major cell-surface receptor of HA. CD44 has many isoforms of different molecular weights. The standard CD44 molecule ranges between 85 and 95 kDa [30]. To examine whether the 85-kDa HABP was CD44, we treated the COC extract after cultivation with a specific antibody to CD44 to immunodeplete CD44 before ligand-blotting analysis using the FITC-HA method (Fig. 3). The 85-kDa HABP was not depleted in the samples treated with mouse IgG purified from normal mouse serum (Fig. 3, lane 2). Incubation of the COC extract with an anti-CD44 monoclonal antibody resulted in depletion of the 85-kDa HABP (Fig. 3, lane 3).



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  		FIG. 3. Identification of the 85-kDa HABP in porcine COCs. The CD44 was immunodepleted with its specific monoclonal antibody from the COC extracts, and then the supernatant was applied to ligand-blotting analysis using the FICT-HA method (lane 3). As a control, the COC extracts untreated with immunodepletion (lane 1) and treated with purified mouse IgG from normal mouse serum (lane 2) were used. The arrowhead indicates the 85-kDa HABP

Behavior of CD44 in Cumulus Expansion During IVM

Figure 4 shows the process of cumulus expansion and change in CD44 expression in porcine COCs during IVM. The degree of cumulus expansion increased gradually until 48 h in culture (Fig. 4A). Western blotting analysis indicated the porcine COCs clearly expressed the standard isoform of CD44 and the level of its expression increased corresponding to the degree of cumulus expansion (Fig. 4B).



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  		FIG. 4. Behavior of CD44 in cumulus expansion during IVM. A) Morphological changes of the degree of cumulus expansion. Bar = 500 µm. B) Western blotting analysis of CD44. The extracts of COCs before culture (lane 1), after culture for 24 h (lane 2), and after culture for 48 h (lane 3) were analyzed by Western blotting. The arrowhead indicates CD44

Localization of CD44 in Porcine COCs

To determine the localization of CD44 in porcine COCs, the COCs were fractionated. Figure 5A shows the results of Western blotting analysis with anti-CD44 monoclonal antibody. In the COCs, CD44 appeared in the cumulus cells extracts (Fig. 5A, lane 3) but not in the oocyte extracts (Fig. 5A, lane 2). To further examine the cellular localization of CD44 in the COCs, we examined the distribution of CD44 by cell fractionation analysis. As shown in Figure 5B, CD44 was detected exclusively in the membrane fraction, and no band was detected in other fractions. In immunofluorescence analysis, every tomogram and the tridimensional reconstruction of the confocal sections also demonstrated the same localization of CD44, confirming the biochemical evidence (Fig. 6).



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  		FIG. 5. CD44 localization in the porcine COCs. A) The extracts of COCs after culture for 48 h (lane 1), oocytes (lane 2), and cumulus cells (lane 3) were analyzed by Western blotting. The arrowhead indicates CD44. B) Analysis of CD44 localization by cell fractionation. COCs were separated into four fractions using a cell fractionation technique. Lane 1: nuclei and cell debris fraction; lane 2: cytoplasm fraction; lane 3: membrane fraction; lane 4: insoluble fraction. The arrowhead indicates CD44



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  		FIG. 6. Immunofluorescence localization of CD44 in porcine COCs. A) The immunolocalization of CD44 (green). B) The nucleus stained with propidium iodide (red). C) Merged image of A and B. D) Tridimensional reconstruction of the confocal sections. Bar = 50 µm


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we demonstrated the expression of HABPs in porcine COCs during cumulus expansion. Our results clearly showed that many HABPs exist in porcine COCs and that their expressions may be involved in the function of HA with respect to formation of the extracellular matrices of COCs during cumulus expansion.

In previous studies, to examine the activity and localization of HABPs in cell cultures and tissue sections, HA-binding assays using ligand blotting of tissue and cell extracts have been used. In many of these studies, labeled HA was used as a probe to detect HABPs in many tissues [31–35] but not in the COCs. In this study, to detect HABPs in porcine COCs during cumulus expansion, we applied two protocols using FITC-HA or bHA as a probe. Both methods were able to detect HABPs. However, the sensitivity of the bHA method was comparatively low, and nonspecific reactions were observed in the negative control. On the other hand, the FITC-HA method was less time consuming and had better sensitivity and specificity than the bHA method. From these experiments, we concluded that the FITC-HA method is better for detecting HABPs in porcine COCs.

We applied the FITC-HA method to porcine COCs during IVM, and the results indicated that the expression pattern of HABPs changed during culture. Of the HABPs detected, the 85-kDa HABP was, interestingly, detected only after 48 h in culture, although other HABPs could not be specified or identified in this study. This finding suggests that the 85-kDa HABP is probably essential for cumulus expansion and oocyte maturation, as the present conditions for in vitro culture could help most COCs in the pig to undergo cumulus expansion and also oocyte maturation within 48 h [36]. In the experiment to identify the 85-kDa HABP, immunoprecipitation analysis showed that this HABP was likely CD44. CD44 is the principal cell-surface receptor for extracellular matrix HA and exists in a number of isoforms with different molecular sizes (80–250 kDa) on a wide variety of cell types [37–40]. CD44 has important functions in several pathophysiological processes such as inflammation and metastatic spread of cancer cells [41, 42]. Like these, the form and function of CD44 can change dependent on cell type. However, the molecular size and function of CD44 expressed in the porcine COCs have not been determined. The results of the present study indicated that CD44 expressed in the porcine COCs had its molecular size range in the standard form. Moreover, we demonstrated that the expression of CD44 in porcine COCs increased in a manner dependent on the degree of cumulus expansion. Therefore, these results suggest that CD44 is correlated with the retention of HA in the extracellular space of the porcine oocytes as the cumulus expands during oocyte maturation. This correlation might be observed not only in vitro but also in vivo. There is less information on the expression of CD44 in COCs in vivo. However, considering the expanded COCs and the presence of HA matrix around the cumulus cells in preovulatory follicles after gonadotropin stimulation [43], a similar CD44-behavior might be seen in vivo.

In the last several years, a great deal of interest has been focused on the signal transduction of HA [44–47]. At present, however, such signal transduction of HA in expanded COCs is still unclear. Recent studies have indicated that proteins derived from serum are essential for retention of HA in expanded COCs [48–50]. As HABPs can be grouped according to their extracellular and subcellular distributions [51], these serum proteins seem to belong to a group of extracellular HABPs. Thus, it appears that such proteins are not able to participate in the signal transduction of HA in expanded COCs. Recently, it was demonstrated that CD44 also functions as a signaling receptor in a variety of cell types [46, 52]. The present study indicated that CD44 is expressed on/in the membrane of cumulus cells. This finding is consistent with the results of the previous study, which demonstrated the expression of CD44 mRNA in cumulus cells but not in oocytes of pigs by reverse transcription-polymerase chain reaction [23]. Considering the existence of CD44 in the membrane of cumulus cells, we speculated that CD44 may be involved not only in the retention of HA in the extracellular matrix but also in communication with cumulus cell-matrix interactions as a signaling receptor in porcine COCs during oocyte maturation. Although there has been no study in which the necessity of HA for the porcine oocyte maturation has been clearly demonstrated, there are many reports of the relation between cumulus expansion and oocyte maturation. Since the main component of cumulus expansion is hyaluronic acid, it suggests the close correlation between HA and oocyte maturation. Therefore, elucidation of the signal transduction of HA-CD44 interactions in the COCs may be a key to understanding the mechanism of oocyte maturation.

In summary, the present study indicates that ligand blotting with FITC-HA is adequate to detect HABPs in porcine COCs and that the level of expression of 85-kDa HABP increases with cumulus expansion during IVM. In addition, the 85-kDa HABP, which was identified as CD44 by immunoprecipitation analysis, was newly expressed after culture and was distributed only on/in the cumulus cell membrane. These results suggested that CD44 probably forms a network in the extracellular space of the porcine oocyte with cumulus expansion during IVM. Whether the interplay between HA and CD44 is acting on oocyte maturation, as we have hypothesized, deserves additional investigation.


   ACKNOWLEDGMENTS
 
We are grateful to the staff of the meat inspection office, Sendai city, for supplying porcine ovaries.


   FOOTNOTES
 
1 Program for Promotion of Basic Research Activities for Innovative Biosciences; grant sponsor: "Research for the Future" program, the Japan Society for the Promotion of Science; grant JSPS-RFTF97L00904. Back

2 Correspondence: Masaki Yokoo, Laboratory of Animal Reproduction, Graduate School of Agricultural Science, Tohoku University, 1-1 Tsutsumidori-amamiyamachi, Sendai 981-8555, Japan. FAX: 81 22 717 8687; myokoo{at}bios.tohoku.ac.jp Back

Received: 22 February 2002.

First decision: 17 March 2002.

Accepted: 6 May 2002.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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